Thalassemia: The consequences of unbalanced hemoglobin synthesis

Thalassemia: The Consequences of Unbalanced Hemoglobin Synthesis* DAVID

G.

NATHAN,

M.D.

and ROBERT

B. GUNN,

M.D.

Boston, Massachusetts

T

HALASSEMIA major or Cooley’s anemia is a severe, usually fatal form of inherited anemia. Beginning approximately two to four months after birth, the disease is associated with profound anemia, jaundice, splenomegaly, expanded marrow space, siderosis and cardiomegaly. The peripheral smear is characterized by extreme poikilocytosis, anisocytosis and anisochromia. Target cells and nucleated erythroid precursors abound. Transfusion therapy is mandatory in most cases. Splenectomy may be necessary if marked splenomegaly causes destruction of platelets, white cells or transfused red cells, but the basic anemic process is usually unchanged by the procedure. The devastating clinical effects and the fascinating genetic and pathophysiologic aspects of this severe disease and the various “thalassemia syndromes” [I] have led to multiple probes of these disorders by geneticists, molecular biologists and physicians. The following discussion of thalassemia has as its chief purpose a re-emphasis of the morphologic, erythrometabolic and erythrokinetic abnormalities which attend the disease. Severe thalassemia is less a disorder of depressed hemoglobin synthesis and more a disorder of unbalanced hemoglobin synthesis. Indeed, it is our view that the untoward sequellae of thalassemia are less due to underproduction of normal hemoglobin than to overproduction of aberrant hemoglobin. To support these remarks it is necessary to present a brief review of the genetic basis of the common thalassemia disorders. GENETICS SYNTHESIS

The amino acid composition of these subunits has been established. To each subunit a heme ring is attached. Three other hemoglobins are present in smaller amounts in normal hemolysates. Hemoglobin F comprises less than 1 per cent of normal adult hemolysates and is composed of two alpha and two gamma subunits. Hemoglobin A2 comprises less than 3 per cent of the hemoglobin of a normal hemolysate and is formed by two alpha and two delta chains. Hemoglobin As (approximately 10 per cent of the total hemoglobin) has the same subunit composition as hemoglobin A and is probably a slightly denatured derivative of hemoglobin A. In addition, glutathione is bound in disulfide linkage probably at the site of the thiol groups of the Aa beta chain. Aa is found in aged red cells and is considered part of the A fraction in any clinical or genetic analysis of hemoglobin heterogeneity. (For detailed reviews, see [ 7-31.) In the past decade it has been recognized that there are four homologous pairs of loci concerned with the production of the subunits of globin in normal human erythroblasts. These loci, termed alpha, beta, delta and gamma sites may be jointly or severally affected by a thalassemia lesion which leads to reduction of the net output of a subunit polypeptide. For simplicity we refer to the term beta thalassemia to define suppression of beta subunit production. Thalassemia major or Cooley’s anemia, for example, is now thought to be due to heritable reduction of the output of both loci governing the synthesis of the beta chains of hemoglobin A. Thalassemia minor, or Cooley’s trait, is due to reduction of the output of only one beta locus. These concepts have been widely accepted and provide a working basis for the explanation of many of the phenomena observed in thalassemia. The exact nature of the suppression of the

OF HEMOGLOBIN AND

THALASSEMIA

The major component of a normal hemolysate (hemoglobin A) is comprised of two pairs of homologous subunits, alpha and beta chains.

* From the Hematology Research Laboratories of the Peter Bent Brigham Hospital and the Children’s Hospital This study was supported by U. S. Public Health Service Grant AM-00965, Medical Center, Boston, Massachusetts. and the John A. Hartford Foundation, Inc. VOL.

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net output of a locus is unknown. Several possibilities exist. There may be loss of the section of the chromosome which bears all or part of the locus which is responsible for the encodement of the messenger RNA for one of the subunit polypeptides. The loss may be due to deletion or to crossing over. In support of the latter mechanism are the findings regarding the Lepore trait [4,5] and the hemoglobin Pylos syndrome [6] which are forms of beta thalassemia trait (one beta locus involved) with an accompanying abnormal hemoglobin. The abnormal hemoglobin is comprised of a normal alpha but a distinctly abnormal “beta” chain. The beta chain has been found to be an amalgam of pieces of beta and delta chains. A reasonable cause of this phenomenon would be crossing over with insertion of a piece of delta site into the midst of a beta site. On the other hand, were crossing over to be the most common cause of loss of beta chain production, more cases of the nature of Lepore trait and Pylos syndrome might be expected. In the past few years the Jacob-Monod model [7] has been invoked to provide a more satisfactory explanation for depressed beta subunit accumulation. On the basis of this model it is proposed that an altered repressor of the operator which controls the output of the structural beta subunit locus provides the basis of the disorder. Proof of such a model in human erythroblasts has not as yet been obtained. Another theory explores the possibility that the relatively stable messenger RNA [8] of human reticulocytes might become unstable. A marked instability of beta chain messenger due to the inherited production of an as yet undetermined substance could be responsible for deficient beta chain production. Itano (21 and Ingram and his associates [9] have suggested that thalassemia may be due to a single base substitution within a given triplet of the beta locus. Epstein [70] has recently examined the concept of a single base substitution in mutations, a review to which the reader is referred. Such a substitution would produce a template or messenger RNA which in turn would encode for the same amino acid found in the normal beta chain but be necessarily attended by a transfer RNA which might be produced in much smaller quantity than is the transfer RNA necessary for the usual triplet This new template RNA would sequence. occupy space on the ribosomes and block the ribosomal adhesion of normal RNA. This theory

Gum

is extremely attractive since it would permit many different sites of delay in the assembly of beta chains and would therefore partially account for the variable severity of Cooley’s anemia It is of interest that normal beta chain production is associated with a delay point near the ninetieth amino acid residue whereas no delay points are associated with alpha subunit assembly [77]. On the other hand, the theory does not by itself account very well for the rise in fetal and A2 hemoglobin observed in Cooley’s anemia. Another possible genetic lesion which might occur in thalassemia is specific ribosomal injury. Such an injury might also induce a single base change in messenger RNA of the type envisaged by Ingram and his associates. Streptomycin has been shown to injure cell ribosomes in a fashion which alters single base sequence enough to lead to a “mis-sense” code and amino acid substitution within polypeptides [ 721. The studies of Bank and Marks [ 731 provide no support for such ribosomal damage in thalassemia. A fundamental and fortunate accompaniment of the decreased beta chain production in Cooley’s anemia is an associated rise in fetal hemoglobin. Several theories have been proposed for the persistence of gamma chain production in Cooley’s anemia, but no satisfactory reason for this fortunate circumstance has been established. The concentration of fetal hemoglobin may range from 10 to over 90 per cent of the total hemoglobin present in the peripheral blood. It is important to realize that the cellular distribution of this fetal hemoglobin in Cooley’s anemia is different from that observed in the relatively harmless anomaly, “hereditary persistence of fetal hemoglobin.” In the former the fetal hemoglobin is distributed heterogeneously, whereas in the latter each cell has very nearly the same content of fetal hemoglobin. CLINICAL

VARIETIES

OF BETA

THALASSEMIA

Full expression of clinical severity of thalassemia (thalassemia major) requires that the patient inherit an abnormal allele from each parent. In thalassemia minor only one abnormal allele is inherited and the disease much less severe. The heterozygous or trait forms of thalassemia are usually accompanied by moderate hypochromia and anisocytosis. In the trait form of “classic” (AZ) thalassemia a distinct rise in A2 and a small increase in F hemoglobin is usually observed [74]. In F thalassemia trait, hemoglobin F is increased and hemoglobin AhlEI:,CAN

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Thalassemia-Nathan, A2 is normal. In a few cases both are distinctly elevated. Splenomegaly is either mild or absent, and the disease is well tolerated. The red cell life span is normal or nearly normal [75]. Jaundice is rare and when present [76] may mainly be due to ineffective erythropoiesis. The blood smear resembles that of iron deficiency except that a greater degree of poikilocytosis usually exists at a given hemoglobin level. Since thalassemia trait leads to suppression of one of the two sites of beta subunit production, doubly heterozygous subjects with combined thalassemia minor and a beta-chain hemoglobinopathy trait present a predictable picture. The production of normal beta subunits is markedly curtailed by two different abnormalities affecting the two beta production sites. Therefore, little normal hemoglobin is produced. Accordingly, a higher proportion of the hemoglobin that is produced is of the beta hemoglobinopathic variety. Most cases of thalassemia major and thalassemia minor may be easily distinguished from each other but there is a small group of patients with disease of intermediate severity. For example, the union of two patients with thalassemia minor, one with increased AZ production and the other with increased F production, may produce normal children, children with either AZ or F thalassemia trait or children with moderate anemia, jaundice and splenomegaly and over 90 per cent fetal hemoglobin [ 771. The latter group of patients seem to be double heterozygotes for two different beta thalassemic lesions. These patients often live moderately comfortably into adult life. In some cases parents with thalassemia minor of the high AZ variety may produce children with all the stigmata of thalassemia major including jaundice, splenomegaly and moderately severe anemia. But for some unknown reason these children seem to weather the storm of growth and adolescence although they emerge with clear-cut and, thalassemia major, they have stable hemoglobin values as high as 9 to 11 gm. per cent and may have highly productive lives. Therefore, it behooves the physician to be optimistic in his care, with the hope that a given affected child will be one of these so-called “thalassemia intermedias.” Just as the homozygous forms are sometimes subclassified as thalassemia major and inter-media, the heterozygous forms may be subclassified as thalassemia minor and minima. The terms are useful and self-explanatory. VOL.

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Gunn DELTA

AND GAMMA

THALASSEMIA

Although the terms Cooley’s anemia and Cooley’s trait have been applied principally to the beta thalassemia syndromes it may be considered that the existence of alpha and delta subunits of hemoglobin represents a physiologic form of thalassemia in man since the product of the delta subunit gene is formed at only onethirtieth the rate of the beta chain. On rare occasions complete absence of delta subunit production may be noted [78]. Obviously there are no clinical sequellae of this abnormality. The gradual suppression of the output of gamma chains observed during the maturation of the normal infant might be considered a form of gamma thalassemia. ALPHA

THALASSEMIA

Three important clinical syndromes result from depression of production of alpha chains. Alpha thalassemia trait, presumed depression of the net output of one of two alpha subunit sites, is similar in certain respects to beta thalassemia trait. Mild microcytosis, erythrocytosis, poikilocytosis and hypochromia are present, with microcytosis usually being the most prominent finding. There is no enhanced production at the gamma or the delta site. Consequently, no increase in AZ or F hemoglobin is detected in adult hemolysates. In fact the total amount of AZ hemoglobin may be reduced. The most important disorder to be differentiated from alpha thalassemia trait is that of iron deficiency. The latter is ruled out most definitively by examination of appropriate stains of marrow. Italians, Greeks, Chinese and Negroes are the most commonly affected groups. When combined with a beta hemoglobinopathy such as sickle hemoglobin, alpha thalassemia trait does not lead to an increased percentage of the abnormal hemoglobin in the hemolysates. In fact, the percentage of abnormal hemoglobin may be somewhat lower than that usually observed. In the circumstance in which it is combined with a hemoglobinopathy affecting the trans-alpha site (hemoglobin I) an increased percentage of the abnormal hemoglobin does occur. When matings of two afflicted subjects occur, the result of such a union may be a patient with homozygous alpha thalassemia. In this fatal disease alpha subunit production is either markedly curtailed or totally absent. Gamma chain production does occur and the resulting soluble hemoglobin is Bart’s hemoglobin, a tetramer of

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gamma chains. Bart’s hemoglobin has virtually no Bohr effect and has the additional unhappy ability to bind oxygen nearly irreversibly at the oxygen tensions of the tissues [79], hence conducts the function of oxygen transport extremely poorly. The result is that such infants are usually born dead or dying at approximately the thirtysecond week of pregnancy bearing all the evidence of severe hydrops fetalis whether anemia is severe or moderate. Extreme macrocytosis, anisocytosis and anisochromia, with marked erythroblastemia, are present. The liver is huge, the spleen remarkably small. It should be pointed out that Bart’s hemoglobin may be quite easily detected in the hemolysates of infants with the alpha thalassemia trait, but it disappears when normal gamma chain production virtually ceases and is not routinely detectable in the hemolysates of adults with pure alpha thalassemia trait. When highly sensitive technics are used, Bart’s hemoglobin may be detected in a large proportion of the hemolysates of premature and newborn infants who have no familial evidence of alpha thalassemia [ZU]. This implies that gamma chain production normally outstrips alpha chain production during fetal life, and that the small gamma chain excess forms Bart’s hemoglobin. Another form of alpha thalassemia syndrome is hemoglobin H disease. This disorder also occurs in the family setting of alpha thalassemia trait, but probably cannot be regarded either as a severe form of alpha thalassemia trait or a mild form of homozygous alpha thalassemia. The disease resembles thalassemia intermedia. All the signs of hemolysis are present together with evidence of a disorder of hemoglobin production. When hypochromic hemoglobin H blood samples are exposed to a redox agent such as brilliant cresyl blue, characteristic robin’s egg blue fine precipitates appear within nearly all the cells. The larger cells appear to have more precipitates. Starch block electrophoresis of hemolysates at pH 7 reveal that the hemolysates contain an abnormally rapid fraction which may include from 5 to 25 per cent of the total and which has been shown to be comprised of beta subunit tetramers. This so-called hemoglobin H has several unusual properties. Like hemoglobin Bart’s, it is a useless respiratory pigment. Its lack of Bohr effect and its high affinity for oxygen inhibits its capacity to deliver oxygen to the tissues at physiologic pH or oxygen tension [27]. Therefore, as noted by

Gunn

Gabuzda [22], hemoglobin H traverses the circulation “forever in unhappy union” with oxygen. Hemoglobin H is an unstable tetramer and is readily oxidized within the red cell. It therefore precipitates during the life span of the cell. As it precipitates, it apparently binds glutathione to its thiol groups in a final embrace du mort [22]. The results of the intracellular precipitation of such a substantial quantity of hemoglobin are large intracellular Heinz bodies which may be most easily observed in the peripheral blood of splenectomized patients with hemoglobin H disease. The relationship of Heinz body formation to hemolytic anemia and hemoglobin H disease will be discussed subsequently. The mode of inheritance of hemoglobin H disease is not at all clear. One of the parents of the patient usually has alpha thalassemia trait and the other appears normal. The apparently normal parent seems to carry a defect which permits the expression of hemoglobin H in a child who otherwise might have only alpha thalassemia trait. The defect may be one which depresses the output of the trans-alpha site, or increases the output of the unaffected beta sites. Hemoglobin H disease, then, is a form of alpha thalassemia in which the imbalance between alpha and beta subunit production is greater than that observed in ordinary alpha thalassemia trait. Hemoglobin H may be detected after exposure to brilliant cresyl blue in a small percentage of the erythrocytes of patients with ordinary alpha thalassemia trait. In fact, the detection of these cells constitutes a major clue in establishment of the diagnosis of alpha thalassemia trait. Presumably these rare cells are the progeny of erythroblasts which were so seriously affected by a gross imbalance of beta and alpha production that the excessive beta chains form the tetramer hemoglobin H. The forms of alpha thalassemia serve as a convenient and constructive example of a concept of the pathophysiology of thalassemia which this review intends to illustrate. The basis of this concept is the central role of unbalanced subunit production in the pathogenesis of these disorders; unbalanced production in which the subunit synthesized in excess is as important if not more important in the pathogenesis of disease than is the underproduced subunit. To refine this illustration we must return to a morphologic and erythrokinetic evaluation of Cooley’s anemia. AMERICAN

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Thalassemia-Nathan, MORPHOLOGY

Gunn

AND ERYTHROKINETICS

IN COOLEY’S

ANEMIA

The appearance of the peripheral smear in Cooley’s anemia depends upon the presence or absence of the spleen. When the spleen is intact, there are numerous tear-drop forms. (Fig. 1 and 2.) The red cells are heterogeneous with respect to hemoglobin content and size. Large pale cells and small dense cells may be observed together with intermediate forms. Nucleated red cells are usually present in small numbers. In wet preparations viewed with Nomarski optics, the tear-drop cells may be very striking, and contrast sharply with the cells observed following splenectomy. (Fig. 2.) The acid elution technics of Betke and Kleihauer [23], which define the cellular content of fetal hemoglobin, are somewhat confusing in cases of Cooley’s anemia. Just as the peripheral smear stained with Romanowsky dyes shows marked heterogeneity of hemoglobin distribution so does the Betke preparation. (Fig. 3.) As a result, it may be surmised that some cells contain more fetal hemoglobin than others. Incubation of the Cooley’s anemia erythrocytes of patients with intact spleens in 1 per cent methyl violet at 37’~ for 10 minutes reveals few cells containing Heinz bodies. Measurements of the autologous life span of Cooley’s anemia erythrocytes in patients whose spleens are intact usually reveal biphasic curves. Cr5r survival studies may be more impressive than C14-glycine technics in revealing the fact

FIG. 1. Peripheral smear of a patient with Cooley’s anemia and an intact spleen. Tear-drop forms are evident as well as anisochromia and target cells. Note the teardrop form with a vacuole at its tip. (See text.)

that there is a population of cells which is rapidly removed from the circulation and another with a much longer life span [24]. The glycine-2-Cl4 studies do reveal, however, that the survival of hemoglobin A is considerably shorter than that of hemoglobin F [25]. Therefore, the heterogeneous distribution of hemoglobin F in thalassemic cells has a functional significance, and the cells rich in hemoglobin enjoy a more favorable survival. In splenectomized patients the morphology of the peripheral blood in Cooley’s anemia is markedly different from that observed in nonsplenectomized patients. Tear-drop formation is not as impressive. Instead, there is an increase

FIG. 2. Nomarski optics view of the cells of two patients with Cooley’s anemia; one with an intact spleen (left) and the other splenectomized (right). Note the tear-drop formation in the patient with an intact spleen and the crateral distortions in the splenectomized patient. VOL.

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FIG. 3. Betke-Kleihauer fetal hemoglobin stain of the blood of a splenectomized patient with Cooley’s anemia. The cells rich in fetal hemoglobin are darkly stained. The technic also stains inclusion bodies. It is clear that the largest inclusions are found in the cells with the least fetal hemoglobin. in wide, hypochromic erythrocytes together with small piscine forms which are little more than fragments of stroma. (Fig. 4.) The degree of anisochromia is markedly increased, as is th e number of nucleated red cells. The reticulocyte count is elevated to a lesser extent than in other hemolytic anemias, whereas there may be marked increases in the nucleated red cell count. Incubation of such cells in 1 per cent methyl violet for 10 minutes reveals that many of the wide pale cells contain large Heinz bodies [26].

Gunn

(Fig. 5.) The Heinz bodies are more irregularly shaped and larger than those observed following incubation of normal or glucose-&phosphatase dehydrogenase (GbPD)-deficient cells in phenylhydrazine. Examination of wet preparations of the cells with Nomarski optics [27] shows that the cells have large crateral distortions which may be either indentations of the surface or vacuolar distortions. (Fig. 2.) Electronmicrographs of shadow casts of the cells confirm that the surface is indeed irregular and indented either by vacuoles into which the membrane invaginates, or by actual pits on the surface. (Fig. 6.) Sections of the cells (Fig. 7) examined with the electron microscope also reveal surface indentations, vacuole formations and inclusions of precipitated hemoglobin and degenerated mitochondria. Vacuole formation may also be observed in splenectomized patients with other forms of hemolytic anemia and reticulocytosis [28]. Studies of the survival of the hemoglobins in a splenectomized thalassemic subject have revealed an extremely rapid turnover of a pool of hemoglobin A. In one case, it was shown that this pool represented as much as 50 per cent of the total circulating pool of hemoglobin A and that the turnover of the pool proceeded with a half-time of one day. The turnover of hemoglobin F was considerably slower [25]. Studies of total heme turnover with glycine-2-Cl4 in splenectomized patients have also demonstrated a component undergoing rapid, early destruction 1291. Thus, the morphology and erythrokinetics of Cooley’s anemia suggest an heterogeneous distribution of hemoglobins A and F and further suggest that the large spleen of these patients specifically sequesters those wide, pale cells which contain Heinz bodies and crateral membrane distortions but which possess little hemoglobin F. These studies lend further documentation to the theory that the cells that are poor in hemoglobin F are more liable to destruction. The following data further support this concept. MORPHOLOGY

AND

OF CENTRIFUGED ANEMIA

FIG. 4. Peripheral smear of a splenectomized patient with Cooley’s anemia. Tear-drop cells are not as evident. There is marked anisochromia and anisocytosis.

METABOLISM COOLEY’S

ERYTHROCYTES

When the blood of splenectomized thalassemic patients is centrifuged at 15,000 r.p.m. for 1 hour, the top and bottom 10 per cent of erythrocytes are found to be quite different in character. The upper layer cells are predomiAMERICAN

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FIG. 5. Top (left) and bottom (right) layers of the centrifuged blood of a splenectomized patient with Cooley’s anemia. The top layer cells (1 and 2) are larger and more hypochromic. They also contain larger irregular Heinz bodies and larger crateral indentations than the bottom layer cells (3,4).

FIG. 6. Platinum carbon shadow cast of the membrane as in Fig. 5). Note the numerous surface indentations. VOL.

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of the splenectomized

patient (same

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FIG. 7. Electron micrograph of a section of glutaraldehyde fixed erythrocytes of a splenectomized patient with Cooley’s anemia. Note surface indentations, degenerated mitochondria in vacuoles and Heinz bodies.

nantly newly formed erythrocytes. Reticulocytes are present in much higher proportion in the upper layer. If Fe59 is administered to the patient one week before the centrifugation study, nearly all the radioactivity is found in the upper layer. In time the radioactivity leaves the upper layer

RESULTS

OF

and is found nearer the lower layer of cells. The morphology and hemoglobin composition of the upper and lower layers also differ. The volume and diameter of the upper layer of cells is much greater. Heinz bodies and siderotic granules are larger and more frequent. (Fig. 5.) The hemo-

TABLE I 2 HOURINCUBATIONSOF THALASSEMIC,HEMOGLOBIN H, ABNORMALAND MYELOFIBROTIC GLUCOSE AND AT pH 7.5 PLASMA AT 37 OC. WITH ADDED

Erythrocyte

Thalassemia Whole blood Top layer Bottom layer Hemoglobin H Whole blood Top layer Bottom layer Normal Myelofibrosis

Reticulacytes (%)

10 3

12 3

Hemoglobin

Hemoglobin

35 20 50 12 25 6

Glucose Consumption (mM/L. cells/hr.)

BLOOD

Potassium Flux (mEq./L. cells/hr.) In Out Net

IN AUTOLOGOUS

ATP Content (PM/L. cells)

5.5 7 3

4 7 3

5 9 3.5

-1 -2 -0.5

946 1,040 800

4.0 3 3.5 1.7 3

3 3 5 1.7 2.5

3.5 3 6 1.7 2.3

-0.5 0 -1.0

1,200 1,400 900 1,500 1,850

+:.2

NOTE: The thalassemic and hemoglobin H blood samples were centrifuged at 15,000 r.p.m. for 1 hour and the top and bottom 10 per cent of cells were suspended in autologous plasma for similar studies. An aliquot was retained for starch block or 1 minute alkali denaturation estimates of hemoglobin H or F. The myelofibrotic blood is included as a control because this particular specimen of blood was from a splenectomized patient with many nucleated red cells. The patient was also iron deficient and the mean corpuscular volume and mean corpuscular hemoglobin closely approximated the blood of the patients with thalassemia and hemoglobin H disease. AMERICAN

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Thalassemia--Nathan, globin content of cells in the upper layer is lower than that in the bottom laver and crateral distortions viewed with Nomarski optics appear much larger and more frequent. (Fig. 5.) Fetal hemoglobin content is much lower than it is in the small dense cells in the bottom layer. (Table I.) When cells from the upper and lower layers are incubated separately in vitro, the membrane defect of the cells in the upper layer, those with less hemoglobin and increased size and number of Heinz bodies, crateral membrane distortions and siderotic granules, is very obvious. The flux of K42 across such cells may be extraordinarily high. Glycolysis and lactate formation are excessive. ATP content is low and often unstable. (Table I) The smaller cells of the bottom layer, those with more hemoglobin (much of it fetal hemoglobin), and smaller and fewer Heinz bodies, crateral distortions and siderocytes, have a much more normal flux of potassium and a lower glycolytic rate. ATP levels, however, are lower than in the cells of the upper layer. These data suggest that the cells with an ability to form gamma chains have a higher degree of membrane integrity than do the cells which have more limited gamma chain production. It may also be suggested that cells rich in fetal hemoglobin are smaller than F-poor cells because the former divide more frequently in the marrow and that the larger volume of the F-poor cells is largely comprised of water. PATHOGENESIS COOLEY’S

OF HEMOLYSIS

ANEMIA-AN

IN

HYPOTHESIS

Fessas [26] has postulated that the Heinz bodies in Cooley’s anemia stem from the unbalanced production of alpha chains in cells lacking sufficient beta chain production to combine with the alpha subunits. Since unpaired alpha subunits are extremely unstable, it seems reasonable that they might precipitate and become the antecedents to the Heinz bodies. A logical extension of this argument is that the Heinz bodies may contribute to membrane damage and hemolysis by some mechanism as yet undefined and, furthermore, that cells capable of increased gamma chain production survive more favorably because they contain smaller and fewer Heinz bodies due to the gamma chains which provide a combining site or trap for excessive alpha chains by forming fetal hemoglobin. (Fig. 3.) VOL.

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FIG. 8. Methyl violet preparation of a bone marrow aspirate of a child with Cooley’s anemia. No Heinz bodies were detectable in the peripheral blood. Note the large dark inclusions one of which may be seen extruding from an opening in the cell.

When the spleen remains in situ it removes Heinz bodies from the cells [30]. In fact, some of the splenic hypertrophy observed may be due to its constant phagocytosis of Heinz bodies [37]. In such patients Heinz bodies are seen only rarely in peripheral smears, although many of the bone marrow erythroblasts and some marrow erythrocytes are full of these inclusions. (Fig. 8.) The Heinz body-laden erythrocytes which do escape from the marrow are quickly engulfed by the spleen. The splenic littoral cells may seize them at a projecting point on the cell surface under which lies the Heinz body [32]. The erythrocyte may be stretched into a tear-drop shape while it is firmly anchored to the littoral cell at the site of the Heinz body. Should the erythrocyte break loose, it would emerge into the circulation as a tear-drop form, leaving the Heinz body to be completely digested by the splenic littoral cells. Occasionally a vacuole, which may have contained a Heinz body, is seen at the tip of a tear-drop cell. (Fig. 1.) In the splenectomized state, tear-drop formation occurs to a lesser extent. Instead wide thin discs bearing Heinz bodies and crateral distortions are permitted to circulate long enough to be observed in the peripheral blood. Jaundice and methemalbumin persist. The crateral distortions may represent areas of the cell membrane through which Heinz bodies have been “pitted” by nonsplenic littoral cells or they may represent vacuoles in which Heinz bodies and mitochondria have degenerated. Figure 6 dem-

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Fro. 9. Prussian blue stain of a smear of a splenectomized patient with Co&y’s anemia. Many of the cells contain small dense siderotic granules. A ring of granules is present around the nucleus of an erythroblast on the far left. FIG. 10. The peripheral smear of a splenectomized patient with hemoglobin H disease. The poikilocytosis, anisocytosis and anisochromia are similar to that observed in splenectomized patients with Cooley’s anemia. The development of surface spicules is common in H disease erythrocytes.

onstrates that both types of inclusions lie in or near vacuoles, and Kent and his co-workers [28] have shown that such vacuoles contain acid phosphatase activity indicative of autodigestive capacity. It should be emphasized that the splenic passage of a cell containing vacuoles is as troubled as that of a cell bearing Heinz bodies since both types of inclusion are observed only after splenectomy. The degenerated mitochondria observed in sections of thalassemic cells (Fig. 7) suggest a defect in ATP metabolism in these young cells. The basic defect in hemoglobin production may be responsible. Failure to generate hemoglobin leads to deposition of siderotic granules in erythroblasts and erythrocytes. (Fig. 9.) These are seen more frequently in the erythrocytes of the upper layer and in the bone marrow erythroblasts where they frequently surround the nucleus in a “ringed sideroblast” form. The iron accumulates in mitochondria, reducing their function and capacity to generate ATP. Accordingly we have observed, as have others, that the ATP content of the cells of patients with thalassemia is lower than that expected on the basis of cell age. This reduction of ATP content is not only due to abnormal mitochondrial function but also to the excessive demands on ATP stores placed by the accelerated cation pump and possibly by other ATP requiring systems such as the fatty acid:phospholipid exchange system [33 1. Although these mechanisms provide a useful

theoretical model for the hemolytic mechanism, complete experimental proof is not yet available. The Heinz bodies have not been proved to be composed of denatured alpha chains. The central role of the Heinz body in the hemolytic state is not established. Feline red cells frequently contain methyl violet-staining inclusion bodies termed Schmauch bodies [34,353, yet cats do not show evidence of comparably rapid hemolysis. Since the Heinz bodies are found in the most hypochromic cells, the hypochromia itself may be responsible for hemolysis. The latter argument is not satisfactory particularly because hemolysis, although usually present, is not a striking aspect of iron deficiency [36 1. In fact, the life span of the iron-deficient cell is considered by some investigators to be normal [37]. Proof of the deleterious effects of Heinz bodies themselves on the survival of human erythrocytes is strongly suggested but is not clearly established at this time. Experimental production of Heinz bodies indeed leads to accelerated erythrocyte destruction, but the erythrocyte membrane is damaged by any procedure which may create hemoglobin precipitation [38,39]. The in vitro incubation and the morphologic studies cited demonstrate that the most severely compromised thalassemic cells are those containing Heinz bodies. In addition cells with Heinz body liability due to glutathione deficiency [40] or to an abnormal hemoglobin such as Ube, Cologne or Zurich [47-431 are all highly susceptible to hemolysis by drugs and, in fact, may be rapidly AMERICAN

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Thalassemia-Nathan, removed even in the absence of oxidant drugs. If Heinz body formation contributes to membrane distortion and dysfunction in Cooley’s anemia cells, similar findings should be identified in the Heinz body-laden cells observed in hemoglobin H disease. MORPHOLOGY,

ERYTHROKINETICS

ERYTHROCYTE HEMOGLOBIN

METABOLISM

AND IN

H DISEASE

The morphology of hemoglobin H disease differs somewhat from that of Cooley’s anemia. When the spleen is intact there are usually fewer tear-drop forms in hemoglobin H disease than there are in Cooley’s anemia, and the cells are more uniform with respect to hypochromia and volume. On the other hand, the cells of splenectomized patients with hemoglobin H disease may be strikingly heterogeneous with respect to size and shape. (Fig. 10.) Erythrokinetic studies with Fe59, Cr5l and glycine-2-Cl4 in hemoglobin H disease differ most distinctly from those of to a paCooley’s anemia. Fe5g administration tient with Cooley’s anemia is followed by rapid transfer of Fe59 from the plasma to the marrow. There is then a slow emergence of only approximately 15 to 30 per cent of the labeled iron from the marrow into circulating red cells. Much of the iron is found in the nonheme iron of the circulating erythrocytes rather than in hemoglobin [44]. These findings are diagnostic of rapid erythroblast proliferation in the marrow, erythroblastic death in situ and a block in hemoglobin synthesis with resultant iron overload. As already described, the life span of the cells which do emerge in Cooley’s anemia depend upon their hemoglobin F content. In hemoglobin H disease there is also rapid disappearance of Fe jg from plasma to marrow, but more than 50 per cent of the Fesg finds its way back to the circulating red cells from the marrow and much of the Fe5g is found in hemoglobin. Thus red cell production is considerably more “effective” in hemoglobin H disease than it is in Cooley’s anemia. On the other hand, the survival of the red cells is much more uniformly diminished in hemoglobin H disease [45]. In fact both C51 and glycine-2-Cl4 survival curves suggest hemolytic anemia with very nearly a single rate constant expressing the destruction kinetics. To be sure, separate analyses of the rates of disappearance of hemoglobins H and A with glycine-2-Cl4 have shown that hemoglobin H disappears more rapidly from the circulation VOL.

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than does hemoglobin A. Cr5l curves are beclouded in this respect because some of the Cr5i label transfers from hemoglobin A to hemoglobin H during the course of the erythrocyte survival [46]. Whether the spleen significantly influences the destruction of hemoglobin in hemoglobin H disease is not entirely clear [46-B]. Further consideration of morphology is required to elaborate on this point. Centrifugation of hemoglobin H disease cells of splenectomized patients at 15,000 r.p.m. for 1 hour reveals two distinct populations. The upper 10 per cent of cells contains few Heinz bodies, but the cells are filled to a variable extent with soluble hemoglobin H. The bottom 1’0 per cent of cells have much less soluble hemoglobin H and total hemoglobin, and in many of the cells there is a single large round Heinz body. (Fig. 11 and 12.) Studies of the metabolism of these layers of cells show that potassium flux is more active in the cells of the bottom layer than in the top layer, and glycolysis and lactate production do not differ greatly in the two layers even though reticulocytes reside in the top layer. (Table I.) In patients with hemoglobin H disease and intact spleens, Heinz bodies are absent. Potassium flux determinations are approximately equal in top and bottom layers. These findings strongly suggest certain mechanisms of hemolysis in a hemoglobin H disease hemolysis. The newly formed cells in hemoglobin H disease emerge from the marrow with variable amounts of soluble p4. This tetramer (hemoglobin H) is more stable than a4 but is not as stable as a$2 (normal hemoglobin). Therefore it precipitates during its life span and forms Heinz bodies which are insoluble. (Fig. 11.) The larger the initial amount of hemoglobin H within the cell, the sooner a Heinz body w-ill develop and the larger it will be. As the Heinz body forms, it contributes to membrane damage, cation flux is increased and the cell beccmes liable to engulfment by the reticuloendothelial system. The spleen, of course, may be chiefly responsible for this removal. Splenectomy does not, however, prevent Heinz body formation and the hemolytic anemia, although somewhat diminished, continues after splenectomy. Many of the cells found in the circulation after splenectomy may be so deformed and devoid of hemoglobin that their presence in the circulation cannot significantly benefit the patient. (Fig. 10.) The crateral malformations seen with Nomarski optics are found as frequently in

826

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Gunn

FIG. 11. The top (left) and bottom (right) 10 per cent of centrifuged erythrocytes of a splenectomized patient with hemoglobin H disease. The dye induces small precipitations of soluble hemoglobin H which are found in top layer cells. The bottom layer cells contain single large Heinz bodies and less soluble hemoglobin H.

bottom layer nonreticulated hemoglobin H cells as in top layer cells. These crateral malformations may be due to “pitting” or degeneration of the hemoglobin H Heinz body throughout the reticuloendothelial system and this in turn may explain why hemoglobin H survives for a shorter period than hemoglobin A. Hemoglobin H disease is thus an excellent example of the deleterious effect of the hemoglobin subunit that is made in excess in the thalassemia syndromes. Were it not for the production of /?4tetramers, this disease would not be nearly as severe. ALPHA:

BETA THALASSEMIA

Perhaps the best illustration of the physiological value of balanced hemoglobin subunit production may be found in a consideration of This syncombined alpha : beta thalassemia. drome in which one alpha locus and one beta locus is affected by a thalassemia lesion has been described by Fessas and his co-workers [49] and more recently by Pearson [50]. There is no imbalance of subunit production in this syndrome. As in Cooley’s anemia and hemoglobin H disease, two of the four subunit loci are presumably affected, but the affected sites are not homologous pairs, therefore no gross evidence of unbalanced production is detected. In fact, the patients are only mildly anemic and hemoglobin synthesis is no more inhibited than in alpha thalassemia or beta thalassemia traits alone. Slight diminution of haptoglobin content

may be observed in such patients, but no other evidence of hemolysis can be found. DIFFERENTIAL TREATMENT

DIAGNOSIS

AND

OF THALASSEMIA

Appreciation of the sequellae of unbalanced subunit production in thalassemia permits the physician to undertake fairly simple diagnostic steps which discriminate Cooley’s anemia and hemoglobin H disease from the various nonthalassemic, refractory iron-loading hypochromic anemias. This group of disorders, the colorful names of which include “refractory sideroachrestic anemia,” “iron loading anemia,” “familial sex-linked hypochromic anemia,” “pseudothalassemia” and “pyridoxine responsive anemia,” have several aspects in common [57,52]. They may occur more often in males and frequently begin in adult life. Splenomegaly is not an impressive feature of the hypochromic microcytic anemia. Pyridoxine administration, phlebotomy, androgens and liver extract may produce partial hematologic improvement. Anemia in these disorders probably results from an acquired or inherited defect in the formation of heme [53]. A search for evidence of unbalanced subunit synthesis has been negative in such patients. To be sure, their erythrocytes and erythroblasts are heavily surcharged with iron granules which frequently surround the nucleus of erythroblasts in the so-called “ringed sideroblast” form [54]. But incubation of erythrocytes

Thalassemiaor bone marrow cells in methyl violet or brilliant cresyl blue does not reveal other abnormal inclusions. Evidences of marked hemolysis are much less obvious in these disorders. Methemalbumin is rarely detected. The morphology of the peripheral smear is not always a helpful discriminating feature. Severe cell distortion, anisochromia and poikilocytosis may be nearly as striking as that observed in Cooley’s anemia or hemoglobin H disease. Tear-drop forms are often present but they are not as numerous as those observed in the thalassemias, perhaps because less Heinz body formation occurs. The mild degree of unbalanced subunit synthesis which characterized the thalassemia traits renders these diagnoses more difficult to establish without electrophoretic examinations. Even electrophoretic studies are of little value in establishment of the diagnosis of alpha thalassemia trait. Lacking adequate data for a complete family study, this diagnosis can be made only by exclusion, unless it can be established in the research laboratory on the basis of differences in rates of incorporation of labeled amino acid into the separated alpha and beta chains of hemoglobin [55-571. The detection of hemoglobin H cells in alpha thalassemia trait depends in large part on the determination of the microscopist. They are exceedingly rare. We have not as yet examined bone marrow smears of patients with known alpha thalassemia trait for such cells. Methyl violet preparations do not reveal Heinz body inclusions in the peripheral erythrocytes of patients with beta thalassemia trait. We have not searched systematically for inclusions in bone marrow although they might well be present in numbers greater than those observed in normal subjects or in patients with, iron deficiency. Iron deficiency is the most common differential diagnostic problem encountered in the evaluations of thalassemia trait. Prussian blue stain of a marrow smear is the most reliable method of evaluating iron stores. The treatment of thalassemia should be directed toward repair of the consequences of unbalanced subunit production. Red cell transfusion is of course the mainstay of therapy, but splenectomy may rid the body of an organ hypertrophied by the work necessitated by the filtration and destruction of Heinz bodies and derormed cells. The result of this hypertrophy may be dangerous platelet or granulocyte sequestration as well as the shortening of the life span of transfused cells and interference with VOL.

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Starch block electrophoresis at pH 7.0 of FIG. 12. hemolyzates of nearly equal hemoglobin concentrations prepared from the centrifuged blood of a splenectomized patient with Hemoglobin H disease. The anode is on the left. l-whole blood, 2-top layer, 3-lower layer. Note that the top layer contains more hemoglobin H (the anodal fraction) than does the whole blood and considerably more than the Heinz body-laden bottom layer.

nutrition. In some cases there is a rise in hemoglobin concentration after splenectomy, but for the most part such increases are not impressive, presumably because the very deformed cells which are removed by the spleen do not contain much soluble hemoglobin, and they are removed rapidly in any case by extrasplenic littoral cells. Cr51 scans of the spleen performed during autologous Cr5r survival studies in hemoglobin H disease may be quite misleading [&I. C$ moves from hemoglobin A to hemoglobin H during the erythrocyte survival study. It therefore labels the Heinz bodies in high concentration. The spleen is the major site of removal of the Heinz bodies when it is present. Since the Heinz bodies comprise only a small fraction of the hemoglobin present in the blood of these patients, a rise in splenic radioactivity disproportionate to the total sequestered hemoglobin is observed. Splenectomy may have disappointing results in such circumstances. Other forms of therapy include folic acid to prevent “relative nutritional deficiency” [58] in

Thalassemia--Nathan,

828

a highly proliferative bone marrow. Iron chelating agents may be employed to rid the body of the excessive iron [59] which may lead to heart failure due to cardiac mitochondrial iron overload. The iron chelating agents presently available have not proved to be exceptionally effective. More rational forms of therapy of thalassemia are to be expected. As methods of isolation and characterization of the components of protein synthesis become more firmly established, extracts containing normal messenger RNA conceivably might be manipulated in such a manner as to replace the alleged defective messenger which occludes the polyribosomes of the thalassemic cell [60]. Bone marrow transplantation technics may become more adaptable to human diseases. Gamma subunit production might be stimulated in a larger number of cells by certain drugs or hormones. Finally, efforts might be profitably directed to suppression of the subunit made in excess, i.e., beta chains in hemoglobin H disease and alpha chains in Cooley’s anemia. In this manner it might be possible to convert thalassemia to a disease more closely resembling iron deficiency. Hypochromic anemia without excessive hemolysis and the presence of full iron stores is relatively well tolerated by children [67]. Drugs which inhibit the synthesis of rapidly produced proteins are currently available, permitting in vitro application of this approach. It is to be hoped that such efforts, directed at the basis of the disease, will be fruitful. SUMMARY

AND

CONCLUSIONS

In this review we have attempted to emphasize the morphology, erythrokinetics and metabolism of the heterogeneous cells observed in thalassemia. In so doing our chief aim has been to make clear that the clinically overt disorder is in many respects due to the fraction of hemoglobin that is made rather than the fraction that is not made. Severe thalassemia cannot be considered to be simply a hypochromic anemia. It is above all a hemolytic and Heinz body anemia associated with pronounced abnormalities of red cell metabolism. Treatment might well be directed to correction of the abnormalities induced by unbalanced subunit production. In fact, it may well be practical to consider therapeutic attempts designed to reduce the production of excessive alpha chain subunits in Cooley’s anemia and of beta subunits in hemoglobin H disease. Future trials of drugs capable of such

Gunn

effects in vitro may well lead to more successful management of these patients than can be accomplished today. Acknowledgment: The electron microscopy and Nomarski optics views illustrated in this review were performed by or under the direction of Doctors Don W. Fawcett and Jean-Paul Revel, Department of Anatomy, Harvard Medical School. REFERENCES

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End of Symfiosium

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