Refer to: Valentine WN: Hereditary spherocytosis revisited-Eighth Annual Paul M. Aggeler Memorial Lecture-Medical Staff Conference, University of California, San Francisco. West J Med 128:3545, Jan 1978

Medical Staff Conference

Hereditary Spherocytosis Revisited Eighth Annual Paul M. Aggeler Memorial Lecture Delivered October 25, 1977 San Francisco General Hospital Medical Center

WILLIAM N. VALENTINE, MD, Los Angeles

These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California, San Francisco. Taken from transcriptions, they are prepared by Drs. David W. Martin, Jr., Associate Professor of Medicine, and Robert C. Siegel, Associate Professor of Medicine and Orthopaedic Surgery, under the direction of Dr. Lloyd H. Smith, Jr., Professor of Medicine and Chairman of the Department of Medicine. Requests for reprints should be sent to the Department of Medicine, University of California, San Francisco, CA 94143.

DR. KREVANS: * I would like to welcome all of you to the annual lecture in the memory of Paul Aggeler, who was an outstanding hematologist and human being. He was a close personal friend of many in this room, and a colleague of mine and many other hematologists. We are privileged this morning to have as the Aggeler Lecturer Dr. William N. Valentine from the University of California, Los Angeles, one of the genuine leaders in the field of academic hematology in the United States today. His published work has ranged from fundamental observations on leukocytes to his recent elegant focus on understanding the erythrocyte. He has made an enormous number of important clinical and fundamental contributions to our understanding of the internal metabolism *Julius R. Krevans, MD, Acting Chief of the Medical Service, San Francisco General Hospital Medical Center, and Dean of the School of Medicine and Professor of Medicine, University of California, San Francisco. tWilliam N. Valentine, MD, Professor of Medicine, University of California, Los Angeles, Center for the Health Sciences.

of red blood cells and the clinical diseases that spring from the disorders of that metabolism. DR. VALENTINE:t Thank you. I am very pleased and honored to be the Aggeler Visiting Professor for this year. I knew Paul Aggeler well and I admired him very much. He was a man who had a degree of quiet humility and self-effacing qualities that really belied his enormous contributions to medicine. I have entitled my discussion, which is really a review, "Hereditary Spherocytosis Revisited."

History and Introduction I have on several occasions endeavored to see what makes up blood. I obtainied a drop of it from my own hand and I saw it wt'as composed of small round globules, driven through a crystalline humidity of water. -ANTON VAN LEEUWENHOEK. Letter. April 7. 1674

Few hematologic diseases have been investigated with more diligence and ingenuity without yielding their ultimate secrets than has hereditary THE WESTERN JOURNAL OF MEDICINE

35

HEREDITARY SPHEROCYTOSIS

spherocytosis (HS). Leeuwenhoek, the father of hematology, considered that mammalian blood contains "red globules," although his sketches of amphibian blood suggest a biconcave discoidal shape. Most 18th century texts of physiology concurred. This generally held view was convincingly refuted, however, in a paper read by Hewson before the Royal Society in 1773.1 Although Leeuwenhoek had surmised that a globular shape was most suitable for circulating in the blood vessels, he had not diluted mammalian blood and hence was unable to see its "globules" separately from each other. Hewson, who is perhaps better known for his remarkable contributions to knowledge of the lymphatic system and the coagulation of blood, conducted experiments in which blood was diluted in its own serum. His predecessors either used undiluted blood or diluted the blood with water, which "dissolved" the red globules. The red globules, properly diluted in serum, were seen not to be spherical but to be flat like a coin. Successive increments of water added to serum suspensions caused the coin-like particles to assume a constantly more spherical shape until finally they "dissolved" and appeared colorless. Thus Hewson unwittingly carried out the first crude osmotic fragility test. In other experiments it was observed that if neutral salts were added to water in exactly the proper proportions, the flat shape seen in serum suspensions was preserved irrespective of the degree of dilution. Stronger solutions of salt "shrivelled" or crenated the particles. In still other studies, Hewson perceived that under some conditions the red particles were capable of fragmentation into smaller entities, which nonetheless retained their red color. It was thus established two hundred years ago that the normal human red blood cell is a discocyte and not a spherocyte, and that it may fragment without losing its hemoglobin. More than a century after Hewson's death a congenital disorder characterized by the presence of spherical erythrocytes was conclusively recognized. Although rather excellent prior accounts of the syndrome had been published,2 3 Minkowski4 is usually credited with the first accurate clinical description. However, 29 years earlier, the Belgian physicians Vanlair and Masius2 had reported "microcythemia," described the spherical shape of the red cells and proposed that the cause of anemia in their patient was an abnormally rapid destruction of the erythrocytes. Shortly after the turn of the 20th century, Chauffard recognized 36

JANUARY 1978 *

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* 1

greatly diminished resistance of the erythrocytes to lysis in hypotonic saline and prominent reticulocytosis5'; as hallmarks of the disease. At about the same time an "acquired" form of hemolytic anemia, also characterized by spherical red cells, was described and became known as the HayemWidal type78 in contrast to the congenital Minkowski-Chauffard form. Although a variety of hemolytic states were mistakenly lumped with these entities, the acquired form undoubtedly consisted largely of cases that would be classified today as Coombs-positive autoimmune hemolytic anemia. In the United States the first case of congenital hemolytic jaundice (there were many other synonyms) was reported in 1910,9 and by the time of Tileston's monograph'0 in 1922 most of the major features of the disorder had been clearly suggested. Jaundice appeared at birth or during childhood and persisted in a fluctuating manner throughout life. As Chauffard had observed, many patients were more jaundiced than sick and many attained an advanced age. The disease often involved three or more generations, and the sexes were equally affected. Some family members were free of disease and, when this occurred, so were their children. Icterus was usually not intense, and there were no signs of biliary obstruction such as itching, bradycardia, xanthomata or a greenish tint to the jaundice. The urine was notably lacking in bile; the stools were heavily colored, contained excess urobilin, and were never acholic. Enlargement of the spleen was the rule. Anemia was usually modest, reticulocytosis was prominent, and there were occasional "crises of deglobulization" as the French termed it. At times, the crises were accompanied by deepening jaundice, fever, pain over spleen and liver, and a rapidly worsening anemia. Occurrence of gallstones at a young age was common. The red cells were osmotically fragile in contrast to their increased osmotic resistance in patients with obstructive jaundice. H1emolysins were almost invariably absent. Splenectomy was being done with "increasing frequency and happy consequences."'10 Following the procedure, a permanent clinical cure was predictable, jaundice rapidly subsided, and anemia and reticulocytosis disappeared. Increased red cell fragility and morphologic abnormalities in the circulating blood persisted, however. In contrast to chronic passive hyperemia in which splenic sinuses were engorged with blood, the congestion in some but not all spleens removed from patients with con-

HEREDITARY SPHEROCYTOSIS

genital hemolytic icterus was confined to the pulp or "cords of Bilroth," while the sinuses lay empty.'0 All this by 1922. Hereditary spherocytosis is usually inherited as a Mendelian dominant trait. However, in the large scale studies of Race" and Young and co-workers,'2 families were observed in which the blood of both parents of affected children could not be distinguished from normal by any available technique. Although spontaneous mutation is a possible explanation, in most such instances it is more likely that the expression of the abnormal gene was below the threshold of detection. Such "variable penetrance" is not uncommon, and a minimally affected parent may transmit a much more active form of the disease to offspring. It is also probable that HS, like many genetically determined diseases, is heterogeneous and not the result of a single mutant gene. Although unproved, the possibility of a recessively transmitted mutation could explain atypical cases. In deer mice a hemolytic disorder essentially identical to human HS occurs as an autosomal recessive trait.'3 Unequivocal homozygosity for HS thus far has not been shown, possibly, though not certainly, because the homozygous state may be lethal as suggested by Race." While earlier workers recognized that minor and major crises could punctuate the course of HS (See Dacie'4 ), detailed studies by Owren"5 focused attention on their pathogenesis. In contrast to the generally held earlier opinion that such crises were hemolytic, it is now generally agreed that in major crises a temporary period of marrow aplasia is a far more common cause. In Owren's study,"5 four patients with crisis were members of the same family, supporting earlier reports (summarized by Dacie'4) of "epidemic" familial crises. During the crisis (Figure 1), hemoglobin fell rapidly, severe reticulocytopenia intervened (0 to 0.3 percent), jaundice abated rather than increased, serum iron levels rose, granulocytopenia and thrombocytopenia occurred, and the bone marrow showed acute hypoplasia. Neutropenia and thrombocytopenia, however, are not always evident. Although the cause of the aplastic crisis (now widely recognized as occurring in a variety of chronic hemolytic states) is unclear, its occurrence at times in "epidemics" strongly supports an association with infection, probably most often of a viral nature. In normal subjects, hematopoiesis probably is also transiently depressed during certain infections, but

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Figure 1.-The reticulocyte count (retics), icterus index, and numbers of marrow normoblasts in the days following onset of "aplastic crisis in a patient with hereditary spherocytosis." (Replotted from Owren's data.'5) Note the early reticulocytopenia, diminution in numbers of normoblasts, and diminished icterus followed by reticulocytosis and sharp increase in marrow normoblasts.

( 1 ) this is more severe in a marrow already laboring to compensate for hemolysis or (2)) depression of production of blood cells is more serious and evident when accompanied by rapid peripheral destruction of erythrocytes. Aplastic crises are self-limited (usually 7 to 14 days). If examinations are not carried out sufficiently early, the resurgent marrow may appear cellular with an increase in young precursor cells. If the patient presents still later during recovery, reticulocytosis may obscure the fact that aplasia and reticulocytopenia were present earlier. Although early students of the disease recognized its hemolytic origin, opinions as to its pathogenesis were varied. Chauffard16 championed the possible etiologic role of hereditary syphilis and tuberculosis. Tileston's monograph'0 detailed the various theories held before 1922. These included postulated hemolysins, despite the fact that antibodies were not demonstrable. Others thought to implicate the hemolytic action of unsaturated fatty acids in serum. Tileston'0 more than 55 years ago concluded, however, that the spleen "is a necessary link for the production of other signs of the disease," and in some way "diminished resistance of the red cells is the leading factor in the causation of hemolytic jaundice" of the congenital type. Although Naegeli17 is usually credited with coining the term "spherocyte," its true origin according to Crosby'8 should be attributed to a monograph on blackwater fever published in 1908.19 The authors, British Army officers asTHE WESTERN JOURNAL OF MEDICINE

37

HEREDITARY SPHEROCYTOSIS W-gx

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signed to India, carefully described the cell and proposed it be designated a "spherocyte." Naegeli, however, did invoke the erroneous dictum that the spherocyte was pathognomonic for hereditary spherocytosis, a concept invalidated many years later by Dameshek and Schwartz.20 They showed that spherocytosis is a feature of certain acquired anemias, as well as of experimental anemias produced by antibodies. In HS, the red cells are small (diameter 6.5 to 7jt instead of the normal 7.7 pu on stained blood films) but abnormally thick (2.2 to 4.7 ,t with normal 1.95 /_) .22 Hence, they stain densely and lack the usual zone of central pallor (Figure 2). The increased thickness compensates for the decreased cell diameter, so that the mean corpuscular volume is usually normal or even increased. Haden21 first clearly indicated the relationship of osmotic fragility to initial cell shape, pointing out that the osmotic fragilities of erythrocytes in a variety of human anemias, as well as in various mammalian species, depend on the ratios of their thickness to their diameters (the thickness index of Von Boros22). He succinctly stated the case as follows: The one fundamental variation from normal in congenital hemolytic icterus is the microspherocytosis. The anemia, jaundice, splenomegaly, reticulocytosis and increased fragility are all secondary to the globular form of the

erythrocyte.2"

Quantitative demonstration that osmotic fragility is indeed a function of shape was provided almost simultaneously by Castle and Daland23 and by Ponder24 in 1937. Soon thereafter it was found 38

JANUARY 1978 *

128 *

1

.%

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Figure 3.-The incubated (24 hours, 370C) and unincubated erythrocyte osmotic fragility curves of a subject with hereditary spherocytosis compared with those of normal controls.

that the increased osmotic fragility of HS cells in fresh blood was accentuated by incubation for 24 hours at 371C.121,25 This became the basis of the more sensitive "incubated osmotic fragility test" often utilized in the diagnosis of cases with mild or doubtful abnormalities (Figure 3). When normal human erythrocytes are incubated without added glucose, a series of metabolic events sequentially takes place. First, glucose and then 2,3-diphosphoglycerate levels fall and then adenosine triphosphate (ATP) depletion occurs. Initially, the erythrocyte, an osmometer26 permitting the passage of water within milliseconds and the passage of anions such as Cl- and HCO-3 only slightly more slowly, retains its relative impermeability to cations. Normally, K+ and Na+ slowly leave and enter the cell, respectively, moving "downhill" in accord with gradients resulting from the high intracellular concentration of K+ and the high plasma concentration of Na+. The correction of these movements requires active pumping of Na+ to the outside and K+ to the inside of the cell. This pumping requires a K+-, Na+- and Mg++-dependent adenosine triphosphatase that is inhibitable by ouabain. A breakdown in the pump inevitably ensues when adenosine triphosphate is depleted secondary to glucose deprivation, and pump failure results in progressive loss of impermeability to cations.

The Pathophysiology of Hemolysis in Hereditary Sphirocytosis In the early 1940's, Ham and Castle27 and Dacie28 studied the volume changes in normal and HS cells undergoing sterile incubation in the absence of added glucose (Figure 4). They found that during the first 24 hours, normal cells swell as a result of a somewhat greater net gain of Na+

HEREDITARY SPHEROCYTOSIS

and water over K+ loss. However, this is followed by cell shrinkage back to or even less than the original volume. Hemolysis does not occur during the period when the mean corpuscular volume is maximal, but only later when cell volumes are equivalent to or less than those present before incubation. As Selwyn and Dacie29 commented, hemolysis under these conditions cannot be related solely to colloid osmotic lysis (although changes in cell cation and water content do occur) but must be due to some type of membrane "contraction" resulting in a decrease in the red cell surface-area/volume ratio. Hemolysis occurs when distention of the membrane ultrastructure permits the egress of first the metabolic intermediates and then the larger hemoglobin molecules. The sequence of events during such incubations is essentially the same for HS and normal cells except that all changes are foreshortened and accelerated when HS cells are

studied.27'28 What, then, accounts for the accelerated changes observed with HS erythrocytes? First, HS erythrocytes are abnormally "leaky" and have an increased permeability to Na+.Y-32 This propensity to accumulate sodium and water requires increased metabolism of glucose to man the pump and maintain cation homeostasis. This, in turn, implies greater dependence on glycolysis to regenerate adenosine triphosphate, an increased metabolic rate to sustain the extra effort of increased sodium pumping and greater sensitivity to glucose deprivation. Everything being equal, metabolic depletion of HS cells should be more rapid than that of similarly incubated normal erythrocytes. Second, metabolically depleted normal red cells do indeed physically lose membrane protein and lipids into the surrounding medium.29'33-35 These occur without loss of hemoglobin and result in (1) an increase in the mean corpuscular hemoglobin concentration and (2) the attainment of a critical hemolytic volume through actual loss of effective surface area from the metabolically depleted red cells. Again, this "normal" process is substantially accelerated in HS cells. The membrane "contraction" prophesied by Selwyn and Dacie29 is due, therefore, to membrane losses to which the HS cell is peculiarly susceptible.'335 Considerable controversy has been generated as to whether increased cation permeability, or the increased susceptibility to membrane budding, is primary in causing the shortened red cell survival in HS. It has been pointed out,.'6 for example,

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Figure 4.-Volume changes and time of hemolysis in normal and hereditary spherocytosis (HS) cells undergoing sterile incubation in absence of added glucose. (Redrawn from figure published by Weed5) Note that observed hemolysis does not correspond to period of maximal increase in red cell volume. The volume changes of HS cells are similar to those of normal erythrocytes but occur much earlier. HCT=hematocrit

that the diminution in osmotic fragility demonstrable in HS red cells of subjects rendered iron deficient by phlebotomy is not accompanied by improved cell survival, a fact suggesting that initial cell shape is not the only determinant of hemolysis. Wiley also reported that HS cells with the "leakiest" membranes paradoxically appear to survive the longest in vivo.37 However, irrespective of the precise role of increased Na+ flux and increased susceptibility of depleted cells to membrane loss in the pathogenesis of hemolysis, the fact remains that both properties represent departures from normal in the osmotically fragile hereditary spherocyte. Normal cells that are metabolically depleted become less plastic and deformable, but freely circulating HS cells are already less deformable than normal. Jandl and co-workers'8 and later others39,40 showed decreased filterability of HS cells in vitro. Leblond and associates41 ingeniously measured the pressure required to deform the membrane or to pull an entire cell into a micropipette whose aperture diameter was smaller than that of the red cell. Hereditary spherocytes had notably reduced deformability, but this applied only to the mature cell. Marrow precusor cells and reticulocytes did not differ from normal by THE WESTERN JOURNAL OF MEDICINE

39

HEREDITARY SPHEROCYTOSIS MUTANT GENE

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MEMBRANE LESION

HEREDITARY SPHEROCYTE

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Figure 5.-Diagrammatic representation of the pathogenesis of hemolysis in patients with hereditary spherocytosis. The spleen provides a hostile environment where the hereditary spherocyte rapidly undergoes progressive changes resulting in its sequestration and destruction.

this technique. Normal red cell deformability has been shown to be highly dependent on adenosine triphosphate and magnesium levels and the maintenance of high adenosine triphosphate to calcium ratios.42'43 The mechanisms by which the spleen plays such an important role in producing the clinical manifestations of HS are closely related to the abnormal properties of the mature erythrocytes. The exact nature of circulation of blood through the spleen is still somewhat controversial. Normal red cells appear to traverse the spleen fairly rapidly, and the transillumination studies of Knisely44-46 supported a "closed" type of circulation, that is, direct continuity between the terminal arterioles and the sinuses. Results of most other studies have supported the concept of an "open" circulation, with many arterioles emptying into the red pulp from which the erythrocytes must find their way back into the sinuses through small apertures in sinus basement membrane. Some of the dilemma of the "physiologically closed, morphologically open" enigma can be resolved if, as Weiss47'48 suggested, the cordal spaces are regarded as partitioned to form a rather complete, if unconventional, vascular channel. Such a channel could conduct blood efficiently, much like an endothelial-lined vessel, from arterial terminations

40

JANUARY 1978 *

128 *

1

to apertures in the sinus wall. Recently, Barnhart and associates,49, 0 as a result of scanning electron microscopy studies on plastic corrosion casts pre-

pared from human spleens, provided evidence of direct arteriovenous shunts and concluded that open and closed types of splenic circulation coexist in man. Be that as it may, the spleen provides a potentially hostile and crowded microenvironment for red cells with defective deformability. The radial arteries in the white pulp give off branches at almost right angles, an anatomic feature resulting in skimming of plasma and fostering the later discharge of concentrated erythrocytes into the red pulp or cordal region. Emerging from here, the red cell (diameter 7 to 8 /t) must traverse apertures in the sinus wall estimated as averaging only 3 , in diameter. In addition, the high endothelium of the smallest splenic arterioles may result in arteriolar apertures sufficiently small as to retard poorly deformable erythrocytes. The splenic test may extend to the sinus itself, where contraction of the terminal end has been observed to result in retention of blood in the sinus lumen for some time.45 The remarkably pliant normal red cell can pass these tests relatively easily, whereas the less pliant HS cells undergo erythrostasis in an almost pasty red cell mass in which glucose is locally depleted, acid metabolites accumulate, and actively metabolizing, phagocytic macrophages abound. Abnormally sensitive to glucose deprivation, abnormally disposed to further membrane losses by budding and lacking in normal deformability, the HS cell undergoes progressive changes leading to its ultimate destruction (Figure 5). Emerson and colleagues25 first transfused serologically identifiable normal donor erythrocytes into recipients with HS scheduled for splenectomy. Studies on the removed spleen showed selective trapping of HS cells, and these were osmotically more fragile than the freely circulating red cells of the patient. Young and associates"l52 confirmed that removed spleens from subjects with HS had trapped the most abnormal erythrocytes. Further, in vitro perfusion of mixtures of HS and normal red cells into spleens removed from patients with idiopathic thrombocytopenic purpura established that these spleens also selectively removed the HS cells. Subsequent investigations",54 with labeled HS erythrocytes have abundantly confirmed their increased propensity to stagnate in the microenvironment of the spleen, where the trapped cells

HEREDITARY SPHEROCYTOSIS

undergo a "conditioning" process. Although initially they may escape back into the general circulation, with each sojourn in an environment predisposed to metabolic depletion there is progressive loss of membrane, progressively less deformability, and the assumption of a progressively more spherocytic shape. After several passages, the spherocyte is no longer able to traverse the tiny apertures leading back into the sinuses, fragments, and undergoes phagocytosis. "Conditioned" cells that have succeeded in escaping a few passages through the splenic circulation account for the most osmotically fragile "tail" characteristically seen on the osmotic fragility curve in hereditary spherocytosis.

The Cellular Defect of Hereditary Spherocytosis What is the actual inherited molecular lesion that represents the ultimate pathogenesis of HS? A variety of abnormal properties of HS red cells have been defined, but none has been identified as the fundamental defect. The two most frequently proposed postulates emphasize an abnorpmality of the red cell membrane but do not specify its nature. As discussed earlier, the first of these emphasizes the predisposition to enhanced membrane loss resulting in reduction of the critical hemolytic volume.55 The second stresses increased permeability to cations, an increase in energy metabolism required by an increased rate of active cation transport and colloid osmotic lysis as the hemolytic mechanism.31'56 Extensive studies of glycolysis in HS compared with normal red cells have included measurements of anaerobic glycolytic and oxidative hexose-monophosphate pathway enzymes, glycolytic intermediates and flux of inorganic P1 into adenosine triphosphate32 and other phosphorylated intermediates of glycolysis.57 Although an occasional study has reported abnormalities, the weight of evidence suggests that no specific abnormality in glycolysis, in the pattern of glycolytic intermediates, or in the flux of P1 into adenosine triphosphate and other phosphorylated compounds exists in HS erythrocytes. Although total lipids have been reported to be somewhat decreased both before33'58 and after35 splenectomy and abnormalities of phospholipid turnover have been suggested,59 no consistently demonstrable data support specific abnormalities in membrane lipid structure. Phospholipids such as sphingomyelin, phosphotidylcholine and phosphotidylserine are reported to lack long chain

fatty acid conjugates (acids with more than 20 carbon atoms), and a reduced capacity of HS membranes for lengthening lipid chains has therefore been postulated.60 Although mature red cells cannot synthesize fatty acids, membrane surface area can be increased and osmotic fragility reduced by media rich in cholesterol or linolenoyl sorbitol.6' Intact HS red cells, as well as their ghost membranes and liposomes prepared from their lipid extract, exhibit increased microviscosity, and this is reported to correlate with the clinical severity of the disorder.6263 Although a role of altered membrane lipids in the pathogenesis of HS remains possible, evidence does not substantiate that observed abnormalities are primary, rather than secondary to the underlying lesion responsible for the morphologic and osmotic abnormalities of the red cell seen even in patients in whom splenectomy has been carried out. Recently, study has focused most frequently on membrane proteins. Lipid-free proteins extracted from HS membranes at low ionic strength resist aggregation when the ionic strength and divalent cation content are increased in comparison with those derived from normal cell membranes. Defective formation of microfilaments in HS cells has been postulated as an explanation.64 Vinblastine at concentrations far higher than those attained when the agent is used therapeutically induces spherocytosis in intact cells and precipitates less protein from HS than normal membranes.65 Vinblastine is known to interfere with microfilament formation in a variety of cells. Agglutination of HS red cells washed in electrolytefree media is reversed only by considerably higher concentrations of added Ca++ than those required for normal erythrocytes.64 Even in normal red cells,42 deformability is altered by changes in the distribution of Ca++, and relative deficiency of a Ca++-dependent adenosine triphosphatase has been reported in HS.66 However, the intcamembrane distribution of Ca++ is not readily ascertained, nor are possible alterations in membrane Ca++ or Ca++-dependent reactions separable into primary or secondary events in HS. Finally, a relationship of abnormally arranged membrane sulfhydryl groups to sphericity is suggested by the fact that agents binding these groups produce sphering of normal erythrocytes. Cells so modified possess a number of properties mimicking those of the hereditary spherocyte.67 Within the past year, several investigations have addressed the concept that altered membrane THE WESTERN JOURNAL OF MEDICINE

41

HEREDITARY SPHEROCYTOSIS

protein phosphorylation may be pathogenetic in HS.08-70 That an abnormal state of microfilamentous membrane proteins having contractile properties may be at fault in HS iS inherently attractive and has been rendered more so by the demonstration that such a protein, membrane spectrin, is a major substrate for phosphorylation by erythrocyte protein kinase. Spectrin is a complex of two peptides and may exist in several aggregation states.7' It resembles myosin (but with some differences), and in vivo it exists as a complex with erythrocyte actin. It interacts with both lipid bilayers and with intrinsic membrane proteins of the red cell, binds Ca-+ and adenosine triphosphate, has distinguishable weak Mg++ and Ca++ adenosine triphosphatase components, and is phosphorylated by adenosine triphosphate in the presence of protein kinase.71 There is evidence that it may play a major role in controlling membrane deformability. Spectrin comprises 20 to 25 percent of the membrane protein, and removal of spectrin from ghosts (red cells without hemoglobin) causes them to break into vesicles. Spectrin is greatly alterable by changes in distribution and amount of red cell Ca++, and normal membrane deformability is maintained only when the cell interior is essentially Ca++-free. In addition, genetically determined HS in mice has been reported to be associated with a severe deficiency of erythrocyte spectrin.72 In short, the search for a defect in the state or function of human erythrocyte spectrin in HS offers an attractive avenue of exploration. Several studies are in agreement that spectrin is the major membrane protein phosphorylated by membrane kinases. Unfortunately, there is little agreement in other respects. Defective phosphorylation of spectrin and of the cyclic adenosine monophosphate-dependent phosphorylation of another membrane protein have been reported by Greenquist and Shohet68 in 22 of 25 patients with HS. Beutler and associates70 concurred with modest diminution in membrane protein phosphorylation of HS cells after 60-minute assays utilizing membrane suspensions. However, the phosphorylation reaction was linear only for short periods, and during the period of linearity HS and normal cells behaved similarly. In contrast, Matsumoto and co-workers69 found diminished spectrin phosphorylation, uninfluenced by previous splenectomy, in HS cells during the nearly linear first ten minutes of incubation. In further support of a role for spectrin, membrane phosphorylation 42

JANUARY 1978 * 128 *

1

was strongly inhibited at the temperature (480 to 50°C) at which spectrin "melts" in a calorimeter.69 Also, red cell sphering began on exposure to concentrations of the sulfhydryl-binding agent, N-ethylmaleimide, which produced similarly decreased membrane phosphorylation in red cell ghosts. The observations are provocative, but a final decision regarding the role of membrane protein phosphorylation in the pathogenesis of HS in man must be deferred. The assay system is very complex, 68-70'73-75 and the exact form of the spectrinactin complex is a complicated function of the ionic strength, status of membrane proteins in the assay material, method of preparation, temperature, time, and distribution and concentrations of divalent cations.71-75 The number of protein kinases potentially involved is unknown. The association of regulator and catalytic subunits in protein kinase is conditioned by a variety of factors, and free and complexed forms of catalytic subunits may even differ radically in their substrate specificity.75 Parallelisms between the effects of heat and sulfhydryl-binding agents on the protein kinase system on the one hand and their ability to produce sphering on the other are complicated by the generalized potential for cellular damage inherent in heating or interfering with ubiquitously distributed sulfhydryl groups of protein. In addition, analogous or even more severe diminutions in membrane-phosphorylating activity than those reported in HS have been described in four cases of hemoglobin sickle cell disease70 and in hereditary stomatocytosis.76 The problem at the moment derives in large part from the inability to know experimentally the spatial relations and composition of enzymes, substrates, cofactors, and divalent cations in highly organized membrane preparations. It is hoped that the discovery of a molecular lesion in HS will evolve from the present base of knowledge, but this happy day has yet to arrive.77

Diagnosis of Hereditary Spherocytosis The diagnosis of HS is usually not difficult. The presence of large numbers of small round densely staining spherocytes on the stained blood film normally reflects either HS or acquired Coombspositive autoimmune hemolytic anemia associated with warm agglutinins. A negative Coombs test in the first instance and a positive test in the second most often serve to establish the correct diagnosis. Confirmation of the spherocytic nature

HEREDITARY SPHEROCYTOSIS

of the erythrocytes is usually readily achieved by means of the osmotic fragility test done on fresh and incubated blood. The increased susceptibility of HS cells to lysis under conditions of glucose deprivation is easily demonstrable by the autohemolysis test of Selwyn and Dacie.28 When normal blood is incubated sterilely and without glucose additives for 48 hours, only minimal hemolysis occurs. When HS blood is similarly incubated, autohemolysis is usually well marked but correctable by glucose additives. Labeling an aliquot of patient cells with chromium 51 and subsequently showing their abnormal sequestration in the spleen by external body counting confirms the phenomenon of splenic erythrostasis, but this is not usually necessary. Further, in many but not all cases of HS, the history of jaundice and anemia from an early age and a family history of early gallstones, jaundice, anemia, splenomegaly and perhaps splenectomy point to its hereditary nature. Normally, this is readily confirmed by family studies that show other affected members and Mendelian dominant transmission. Diagnosis is more difficult in patients with mild abnormalities or when the disorder is not demonstrable in either parent. In the former, differential diagnosis lies chiefly between HS and a wide variety of so-called "nonspherocytic" hereditary hemolytic syndromes such as those associated with certain erythrocyte enzyme deficiency states.

Treatment of Hereditary Spherocytosis The treatment of HS iS splenectomy with the possible exception of fully compensated, very mild cases lacking jaundice, anemia or a history of crises. Even in such cases there is room for argument, because amelioration of mild compensated, but chronic and life-long hemolysis provides some protection against the development of gallstones. The first successful splenectomy was apparently carried out in 1887 by Wells who, operating with the erroneous diagnosis of abdominal tumor, found instead an enlarged spleen.78 Although Wells was unaware of the true diagnosis, Dawson 40 years later restudied the patient and found the characteristically increased osmotic fragility of the red cells in an otherwise healthy patient.78 Wells had done the first splenectomy in England in 1865 and the second and third in 1868 and 1875-all with fatal outcomes. Dawson78 in the Hume Lectures expressed some wonder at the surgeon's temerity in attempting a fourth and some admiration at the operative skill that per-

mitted survival under conditions that did not include asepsis during the procedure. Micheli in 191179 successfully removed the spleen in a patient with the acquired type, and the fortunately brilliant result soon led to widespread use of the procedure with almost complete success in the congenital form of the disease. Splenectomy in HS has limited hazards.80-82 Patients less than 4 to 5 years of age have an increased risk of infection, particularly pneumococcal sepsis, after splenectomy. For this reason, if at all possible the operation should be postponed until the patient is 5 years old. In adults who have had posttraumatic splenectomy, statistical evidence of some slight but significant increase in predisposition to infection has been adduced, and it is said that this is slightly greater in patients with HS.81 However, the risk of serious sepsis is very low. Schilling82 failed to identify a single instance of fatal bacterial infection in a recent retrospective study of 61 subjects with HS (with collectively 800 patient-years after splenectomy). Furthermore, admission to hospital was no more frequent in patients after splenectomy than in normal control subjects. Although splenectomy eliminates anemia and prevents further aplastic crises, its greatest reward is the reduced incidence of complicating cholelithiasis and cholecystitis. Patients with HS in whom splenectomy has not been done should receive folate supplementation daily, a measure that most investigators agree lessens the likelihood of aplastic crises. It should also be mentioned that chronic leg ulcers without obvious underlying vascular causes usually clear after splenectomy in HS patients.83 Occasionally, removal of the spleen is not followed by the usual complete clinical recovery. This has often been attributed to failure to remove accessory spleens or to seeding of the peritoneum by splenic tissue at the time of operation. Cases in which this explanation has been proved are exceedingly rare84 and are convincing only if prompt "cure" results from removal of remaining splenic tissue. Brook and Tanaka85 and also the author have seen cases in which reticulocytosis and jaundice persisted after splenectomy in patients with classic HS who had concomitant heterozygosity for pyruvate kinase deficiency. The speculative possibility exists that associated abnormalities in the glycolytic machinery that are too mild to have clinical manifestations in norTHE WESTERN JOURNAL OF MEDICINE

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mal subjects might result in some diminished survival of the postsplenectomy HS cell with its greater dependence on glycolysis. After splenectomy, certain events normally occur.14 The patient's hemogram returns to normal and remains so. Haptoglobin levels return nearly to normal levels.82 Microspherocytosis persists although often in a less flagrant form because of the absence of the most spherocytic "splenicconditioned" cells. By the same token, osmotic fragility continues to be increased, but the "tail" of most osmotically fragile cells (presumably those with previous passages through the spleen) often disappears from the osmotic fragility curve. Jaundice normally disappears as does significant reticulocytosis, although sensitive measurement of red cell life span indicates that survival is slightly below normal.86 The autohemolysis test also remains abnormal, but in some instances may show improvement in the degree of observed hemolysis. It may be concluded that while much is known about the small, osmotically fragile, densely staining spherocyte, the definitive documentation of the responsible molecular lesion still remains an elusive goal. REFERENCES 1. Gulliver G (Ed): Red particles of the blood, In The Works of William Hewson. Printed for the Sydenham Society, C. and J. Adlard, Printers, 1846, pp 211-236 2. Vanlair C, Masius JR: De la microcyth6mie. Bull Acad R Med Belg 5:515-611, 1871 3. Wilson C: Some cases showing hereditary enlargement of the spleen. Tr Clin Soc London 23:162-172, 1890 4. Minkowski 0: Ueber eine Hereditare, unter dem Bilde eines chronischen Icterus mit Urobilinurie, Splenomegalie and Nierensiderosis verlaufende Affection. Vehr Cong Inn Med Wiesbaden 18:316-321, 1900 5. Chauffard MA: Pathogenie de l'ictere congenital de l'adulte. Sem Med 27:25-29, 1907 6. Chauffard MA: Les icteres hemolytique. Sem Med 28:49-52, 1908 7. Hayem G: Sur une variet6 particuliere d'ictere chronique. Ictere infectieux chronique splenomegalique. Presse med 6:121-125, 1898 8. Widal F, Abrami P, Brule M: Diff6renciation de plusieurs types d'icteres hemolytiques par le procede des hematies deplasmatisees. Presse med 15:641-644, 1907 9. Tileston W, Griffin WA: Chronic family jaundice. Am J Med Sci 139:847-869, 1910 10. Tileston W: Hemolytic jaundice. Medicine- 1:355-388, 1922 11. Race RR: On the inheritance and linkage relations of acholuric jaundice. Ann Eugenics 11:365-384, 1942 12. Young LE, Izzo MJ, Platzer RF: Hereditary spherocytosis; 1. Clinical, hematologic and genetic features in 28 cases, with particular reference to the osmotic and mechanical fragility of incubated erythrocytes. Blood 6:1073-1098, 1951 13. Motulsky AG, Huestis RR, Anderson R: Hereditary spherocytosis in mouse and man. Acta Gent 6:240-245, 1956 14. Dacie JV: The congenital haemolytic anaemias-1. Hereditary spherocytosis, In The Haemolytic Anaemias, Congenital and Acquired-Pt. 1: The Congenital Anaemias, 2nd Ed. New York, Grune & Stratton, Inc, 1960, pp 82-150 15. Owren PA: Congenital hemolytic jaundice, pathogenesis of the "hemolytic crisis." Blood 3:231-248, 1948 16. Chauffard A: Pathog6nie de l'ictere h6molytique congenital. Ann de med 1:3-17, 1914 17. Naegeli 0: Blutkrankheiten und Blutdiagnostik, 3rd Ed. Berlin, Julius Springer, 1923, 560 pps 18. Crosby WH: The pathogenesis of spherocytes and leptocytes (target cells). Blood 7:261-274, 1952

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19. Christopher SR, Bentley CA: Blackwater fever, In Scientific Memoirs by Officers of the Medical and Sanitary Department of the Government of India (N.S.) No. 35, nila, 1908, India (quoted

by Crosby18)

20. Dameshek W, Schwartz SO: Hemolysins as the cause of clinical and experimental hemolytic anemias, with particular refspherocytosis and increased f-ragility. Am J Med Sci 196:769-792, 1938 21. Haden RL: Mechanism of increased fragility of erythrocytes in congenital hemolytic jaundice. Am J Med Sci 188:441-449, 1934 22. Von Boros J: Uber Grosse, Volumen und Form der menschlichen Erythrozyten und deren Zusammenhang. Wien Arch f Inn Med 12:255-272, 1926 23. Castle WB, Daland GA: Susceptibility of erythrocytes to hypotonic hemolysis as a function of discoidal form. Am J Physiol 120:371-383, 1937 24. Ponder E: The spherical form of mammalian erythrocyte; I1I. Changes in surface area in discs and spheres. J Exper Biol 14: 267-277, 1937 25. Emerson CP Jr, Shen SC, Ham TH, et al: The mechanism of blood destruction in congenital hemolytic jaundice. J Clin Invest 26:1180, 1947 26. Guest GM: Osmometric behaviour of normal and abnormal human erythrocytes. Blood 3:541-555, 1948 27. Ham TH, Castle WB: Studies on destruction of red blood cells-Relation of increased hypotonic fragility and erythrostasis to the mechanism of hemolysis in certain anemias. Proc Amer Philos Soc 82:411-419, 1940 28. Dacie JV: Observations on autohaemolysis in familial acholuric jaundice. J Path Bact 52:331-340, 1941 29. Selwyn JG, Dacie JV: Autohemolysis and other changes resulting from the incubation in vitro of red cells from patients with congenital hemolytic anemia. Blood 9:414-438, 1954 30. Bertles JF: Sodium transport across the surface membrane of red blood cells in hereditary spherocytosis. J Chn Invest 36: 816-824, 1957 31. Jacobs HS, Jandl JH: Increased cell membrane permeability in the pathogenesis of hereditary spherocytosis. J Clin Invest 43:1704-1720, 1964 32. Jandl JH: Leaky red cells. Blood 26:367-382, 1965 33. Reed CF, Swisher SN: Erythrocyte lipid loss in hereditary spherocytosis. J Clin Invest 45:777-781, 1966 34. Jacob HS: Membrane lipid depletion in hyperpermeable red blood cells: Its role in the genesis of spherocytes in hereditary spherocytosis. J Clin Invest 46:2083-2094, 1967 35. Cooper RA, Jandl JH: The role of membrane lipids in the survival of red cells in hereditary spherocytosis. J Clin Invest 48:736-744, 1969 36. Crosby WH, Conrad ME: Hereditary spherocytosis: Observations on hemolytic mechanisms and iron metabolism. Blood 15:662-674, 1960 37. Wiley JS: Red cell survival studies in hereditary spherocytosis. J Clin Invest 49:666-672, 1970 38. Jandl JH, Simmons RL, Castle WB: Red cell filtration and the pathogenesis of certain hemolytic anemias. Blood 18:133-148, 1961 39. Teitel P: Le test de la filtrabilte erythrocytaire (TFE): Une method simple de etude de certaines proprietes microrheologiques des globules rouges. Nouv Rev Fr Hematol 7:195-214, 1967 40. Murphy JR: The influence of pH and temperature on some physical properties of normal erythrocytes and erythrocytes from patients with hereditary spherocytosis. J Lab Clin Med 69:758-775, 1967 41. Leblond PF, LaCelle PL, Weed RI: Rheologie des drythroblastes et des erythrocytes dans la spherocytose congenitale. Nouv Rev Fr Hematol 11:537-546, 1971 42. Weed RI, LaCelle PL, Merrill EW: Metabolc dependence of red cell deformability. J Clin Invest 48:795-809, 1969 43. Weed RI, Bowdler AJ: Metabolic dependence of the critical hemolytic volume of human erythrocytes: Relationship to osmotic fragility and autohemolysis in hereditary spherocytosis and normal red cells. J Clin Invest 45:1137-1149, 1966 44. Knisely MH: Microscopic observations on circulatory systems of living transilluminated mammalian spleens and parturient uteri. Proc Soc Exper Biol Med 32:212-214, 1934 45. Knisely MH: Spleen studies; microscopic observations of the circulatory system of living unstimulated mammalian spleens. Anat Rec 65:23-50, 1936 46. Knisely MH: Spleen studies; microscopic observations of the circulatory system of living traumatized spleens and of dying spleens. Anat Rec 65:131-148, 1936 47. Weiss L: The structure of fine splenic arterial vessels in relation to hemoconcentration and red cell destruction. Am J Anat 111:131-179, 1962 48. Weiss L: A scanning electron microscopic study of the spleen. Blood 43:665-691, 1974 49. Barnhart MI, Baechler CA, Lusher JM: Arteriovenous shunts in the human spleen. Am J Hematol 1:105-114, 1976 50. Barnhart MI, Lusher JM: The human spleen as revealed by scanning electron microscopy. Am J Hematol 1:243-264, 1976 erence to the nature of

HEREDITARY SPHEROCYTOSIS 51. Young LE, Platzer RF, Ervin DM, et al: Hereditary spherocytosis: II. Observations on the role of the spleen. Blood 6:10991113, 1951 52. Young LE: Hereditary spherocytosis. Am J Med 18:486-497, 1955 53. Weisman R Jr, Hurley TH, Harris JW, et al: Studies of the function of the spleen in the hemolysis of red cells in hereditary spherocytosis and sickle cell disorders. J Lab Clin Med 42:965-966, 1953 54. Griggs RC, Weisman R Jr, Harris JW: Alterations in osmotic and mechanical fragility related to in vivo erythrocyte aging and splenic sequestration in hereditary spherocytosis. J Clin Invest 39:89-101, 1960 55. Weed RI: Hereditary spherocytosis-A review. Arch Intern Med 135:1316-1323, 1975 56. Jacob HS: The defective red blood cell in hereditary spherocytosis. Ann Rev Mled 20:41-46, 1969 57. Reed CF, Young LE: Erythrocyte energy metabolism in hereditary spherocytosis. J Clin Invest 46:1196-1204, 1967 58. Langley GR, Felderhof CH: Atypical autohemolysis in hereditary spherocytosis as a reflection of two cell populations: Relationship of cell lipids to conditioning by the spleen. Blood 32:569-585, 1968 59. Jacob HS, Karnovsky ML: Concomitant alterations of sodium flux and membrane phospholipid metabolism in red blood cells: Studies in hereditary spherocytosis. J Clin lnvest 46:173-185, 1967 60. Kuiper PJC, Livne A: Differences in fatty acid compositicn between normal human erythrocytes and hereditary spherocytosis affected cells. Biochim Biophys Acta 260:755-758, 1972 61. Livne A, Aloni B, Moses S, et al: Linolenoyl sorbitol and the fragility of hereditary spherocytes. Brit J Haematol 25:429-435, 1973 62. Aloni B, Shinitzky M, Moses S, et al: Elevated microviscosity in membranes of erythrocytes affected by hereditary spherocytosis. Brit J Haematol 31:117-123, 1975 63. Aloni B, Shinitzky M, Livne A: Dynamics of erythrocyte lipids in intact cells, in ghost membranes and in liposomes. Biochim Biophys Acta 348:438-441, 1974 64. Jacob HS, Ruby A, Overland ES, et al: Abnormal membrane protein of red blood cells in hereditary spherocytosis. J Clin Invest 50:1800-1805, 1971 65. Jacob H, Amsden T, White J: Membrane microfilaments of erythrocytes: Alteration in intact cells reproduces the hereditary spherocytosis syndrome. Proc Natl Acad Sci USA 69:471-474, 1972 66. Feig SA, Guidotti G: Relative deficiency of Ca2 *-dependent adenosine triphosphatase activity of red cell membranes in hereditary spherocytosis. Biochem Biophys Res Commun 58:487-494, 1974 67. Jacobs HS, Jandl JH: Effects of sulfhydryl inhibition on red blood cells-1. Mechanism of hemolysis. J Clin Invest 51:779792, 1962 68. Greenquist AC, Shohet SB: Phosphorylation in erythrocyte membranes from abnormally shaped cells. Blood 48:877-886, 1976

69. Matsumoto N, Yawata Y, Jacob HS: Association of decreased membrane protein phosphorylation with red blood cell spherocytosis. Blood 49:233-239, 1977 70. Beutler E, Guinto E, Johnson C: Human red cell protein kinase in normal subjects and patients with hereditary spherocytosis, sickle cell disease, and autoimmune hemolytic anemia. Blood 48:887-898, 1976 71. Kirkpatrick F: Spectrin: Current understanding of its physical, biochemical, and functional properties. Life Sci 19:1-17, 1976 72. Greenquist AC, Shohet SB: Abnormal erythrocyte membrane properties of hereditary spherocytosis in mice. Blood 46: 1005, 1975 (abstract) 73. Zail SS, Van den Hoek AK: Studies on protein kinase activity and the binding of adenosine 3',5'-monophosphate by membranes of hereditary spherocytosis erythrocytes. Biochem Biophys Res Commun 66:1078-1086, 1975 74. Guthrow CE Jr, Allen JE, Rasmussen H: Phosphorylation of an endogenous membrane protein by an endogenous, membraneassociated cyclic adenosine 3',5'-monophosphate-dependent protein kinase in human erythrocyte ghosts. J Biol Chem 247:81458153, 1972 75. Fairbanks G, Avruch J: Phosphorylation of endogenous substrates by erythrocyte membrane protein kinases-II. Cyclic adenosine monophosphate-stimulated reactions. Biochemistry 13: 5514-5521, 1974 76. Mentzer WC Jr, Smith WB, Goldstone J, et al: Hereditary stomatocytosis: Membrane and metabolism studies. Blood 46:659669, 1975 77. Valentine WN: The molecular lesion of hereditary spherocytosis (HS): A continuing enigma (Editorial). Blood 49:241-245, 1977 78. Dawson GE: The Hume Lectures on haemolytic icterusLecture II. Brit Med J 1:963-966, 1931 79. Micheli F: Unmittelbare Effecte der Splenektomie bei einem Fall von erworbenem hamolytischen splenomegalischen Ikterus Typus Hayem-Widal (Spleno hamolytischer lkterus). Wien klin Wchnschr 24:1269-1274, 1911 80. King H, Schumacker HB Jr: Splenic studies; susceptibility to infection after splenectomy performed in infancy. Ann Surg 136:239-242, 1952 81. Singer DP: Postsplenectomy sepsis, In Rosenberg HS, Bolande RP (Eds): Perspectives in Pediatric Pathology, Chicago, Year Book Medical Publishers Inc, 1973, pp 285-312 82. Schilling RF: Hereditary spherocytosis: A study of splenectomized persons. Sem Hematol 13:169-176, 1976 83. Taylor ES: Chronic ulcer of the leg associated with congenital hemolytic jaundice. JAMA 112:1574-1576, 1939 84. Mackenzie FAF, Eastcott HHG, Elliott DH, et al: Relapse in hereditary spherocytosis with proven splenunculus. Lancet 1: 1102-1104, 1962 85. Brook J, Tanaka KR: Combination of pyruvate kinase (PK) deficiency and hereditary spherocytosis (HS). Clin Res 18:176, 1970 86. Chapman RG: Red cell life span after splenectomy in hereditary spherocytosis. J Clin Invest 47:2263-2267, 1968

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