Disorders of Bone Marrow Production

Symposium on Pediatric Hematology

Disorders of Bone Marrow Production Gerald E. Bloom, M.D. *

The bone marrow hypoplasias represent a diverse group of disorders characterized by anatomic defects of marrow function which result in diminished delivery of cells to the peripheral blood. These conditions may involve the formed elements of the blood individually or collectively. With most of the cytopenias, both congenital and acquired causes have been recognized and within each group considerable heterogeneity is apparent. This review will focus attention on the disorders of isolated red blood cell production (hypoplastic anemia) and generalized bone marrow failure (aplastic anemia), since these are the most frequent disturbances of bone marrow production encountered in pediatric practice. Particular emphasis will be placed on newer aspects of diagnosis, treatment, and pathogenesis. Aspects of the conditions which have been reviewed elsewhere recently will not be covered in detail.

HYPOPLASTIC ANEMIAS Acute and chronic forms of hypoplastic anemia have been referred to by a variety of titles including erythroid hypoplasia, red cell aplasia, pure red cell anemia, erythrogenesis imperfecta, are generative anemia, and erythroblastopenia. Collectively, these disorders represent a selective depression of bone marrow erythroid elements without alteration of myeloid precursors or megakaryocytes. In many instances, their causes are unknown, so that the most useful classification is based on clinical characteristics (Table 1). Acute Acute forms of erythroid hypoplasia may persist from a few days to a year before recovery. The development of anemia depends upon the severity of erythroid depression and its duration. With normal red cell sur':'Associate Professor of Pediatrics, and Chief, Division of Pediatric Hematology, University of Florida College of Medicine, Gainesville, Florida This work was supported in part by the Developmental Physiology Training Grant, NIH T1-HD0054. Dr. Bloom is recipient of Career Development Award, No. HD 47446-01.

Pediatric Clinics of North America- Vol. 19, No.4, November 1972

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Table 1. Classification of Anemias Characterized by Diminished Erythropoiesis (Hypoplastic Anemias) I. Acute A. Idiopathic B. Secondary 1. Drug-induced 2. Infections 3. Toxic 4. Marasmus 5. Hemolytic anemia

II. Chronic A. Acquired 1. Idiopathic 2. Associated with thymoma 3. Secondary to systemic disease B. Congenital (Blackfan-Diamond)

vival, a decrease in hemoglobin concentration of approximately 0.7 gm. per 100 ml. per week occurs if erythropoiesis is absent. Therefore, at least several weeks must elapse before anemia can be detected unless additional factors, such as blood loss or hemolysis, are present. The anemia of acute erythroid hypoplasia is characterized by normochromic, normocytic erythrocytes and an absolute reticulocytopenia. Nucleated red blood cells and polychromatophylic erythrocytes are absent. If coexistent nutritional factors such as iron or folic acid deficiency are present, superimposed morphologic evidence of these states may also be evident. Bone marrow examination reveals varying degrees of erythroid deficiency. Occasionally, complete absence of activity is found, although in most instances some evidence of erythropoiesis remains. There is usually uniform depression of all stages of red cell development; however, dyserythropoietic pictures have been described with a predominance of proerythroblasts and occasionally more mature elements. 45 , 181 Overall bone marrow cellularity is normal. IDIOPATHIC. Transient depression of erythropoiesis lasting up to a year may occur in well infants and children without pre-existing hematologic disease. 18o The majority of patients recover spontaneously and recurrences are rare. The etiology of this syndrome is unknown, although the history of a preceding viral-like respiratory illness in some patients suggests an infectious origin. 89 SECONDARY. Drug-Induced. Drug-induced isolated erythroid hypoplasia is unusual. Of 408 cases of chloramphenicol-induced blood dyscrasias recorded by the American Medical Association Registry from 1953 through 1964, only 6 per cent represented selective depression of the red cell series. 9 This was more common in the 20 to 59 year age range than in younger patients. Gasser described 4 patients, ages 11/2 to 13 years, with red cell depression following the administration of penicillin, phenobarbital, or chenopodium. 4'; Adult patients have been reported who developed red cell hypoplasia while receiving sulfathiazol,t60 arsphenamine/ 52 isoniazid,50,lOl tolbutamide,140 diphenylhydantoin sodium/6 or chlorpropamide. 129 Evidence incriminating most of these agents as the

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cause of erythroid hypoplasia is circumstantial. However, direct in vitro data suggesting an etiologic role for diphenylhydantoin sodium in a 17 year old patient were presented by Yunis. 184 Infections. Transient hypoplasia of erythroid precursors probably occurs with relative frequency during the course of common childhood viral infections. Because of its short duration and the relative length of normal erythrocyte survival, clinical evidence of anemia rarely occurs. In certain areas of the world, however, infections may cause red cell hypoplasia and anemia more commonly. In Indonesia, for example, Kho et al. described 238 cases occurring over a 5 year period. 72 Many of these patients had severe overwhelming infections which played a predominant role in the clinical picture. Gasser described acute red cell hypoplasia occurring in association with atypical pneumonia, mumps, and bacterial sepsis. 45 Toxic. In 1963 Schmid et al. reviewed 23 patients with acquired erythroid hypoplasia and added an additional 16}40 Fourteen of the 39 total cases had been exposed to potentially myelosuppressive agents including benzene, pentachlorophenol, insecticides, volatile solvents, dry cleaning fluid, and hexachlorocycloxane. The youngest patient in this group was 34 years of age. Moosa described a well documented case of selective erythroid hypoplasia in a child with lead poisoning. 108 A profound anemia developed and was successfully treated with calcium EDTA. This type of anemia is uncommon in plumbism and is unlike the usual erythropoietic disturbance found in this disorder. 51 Marasmus. Isolated erythroid hypoplasia occurs in about one third of patients with kwashiorkor during the recovery phase. 41 • 176 The anemia is unassociated with iron, folic acid, or vitamin B12 deficiencies and is presumably related to another component of the marasmic picture. Treatment with oral or intramuscular riboflavin has been reported to reactivate the marrows of these patients;41 however, in one group of 46 patients reported by Walt et al.,176 hypoplastic anemia developed in 7 despite the prophylactic administration of 0.75 mg. of riboflavin daily. Hemolytic Anemia. The development of erythroid hypoplasia during the course of chronic hemolytic anemias can lead to disruption of a well compensated hemolytic state. The shortened erythrocyte survival time results in a rapid, at times life threatening fall in hemoglobin concentration when the increased production of erythroid cells is impaired. This complication was first described in congenital spherocytosis120 but may occur in any congenital or acquired hemolytic anemia. 19. 146 Chronic ACQUIRED. Long-standing erythroid hypoplasia in children with previously normal erythropoiesis is uncommon. The disorder occurs more frequently in adult life where 50 per cent of patients have an associated benign thymoma. 133 Although only a single child with a siInilar disorder has been reported,t65 the adult experience is of interest as it indicates that humoral anti-erythropoietic principles may cause selective depression of erythroid precursors in several types of acquired red cell hypoplasia.5. 6. 39. 69. 70. 79. 80. 136. 172

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Chronic erythroid hypofunction in childhood is found most often in patients with a variety of systemic disorders including chronic infections, renal disease, rheumatoid arthritis, hepatic disease, and certain hypoendocrinopathies. 57 In these disorders, decreased erythroid activity is probably related to metabolic alterations associated with the underlying condition, although in none is the mechanism well understood. Various therapies, including iron, androgens, and cobalt, seldom are effective. Rarely is the anemia significantly improved unless the basic underlying process abates. CONGENITAL. More than 100 cases of congenital hypoplastic anemia (Diamond-Blackfan syndrome) have been reported since the disease was described over 35 years ago,27 It is considered to be a primary disorder of the bone marrow, characterized by selective depression of red blood cell production. Symptoms may originate from birth through the first 4 years of life, although the majority of patients present by age 3 months. 28 The anemia is normochromic, normocytic in type and is associated with a reticulocytopenia of less than 1 per cent. At least one developmental anomaly is found in nearly one third of cases. 28 ,105 These include retarded growth, mental retardation, congenital heart disease, digital malformations particularly involving the thumb, and renal abnormalities. Congenital hypoplastic anemia has been reported in multiple ancestral backgrounds, although only two cases in Negroes have been describedY,151 A familial occurrence has been noted by several authors. Burgert 18 described two male siblings who were affected and achieved spontaneous remissions. Atypical familial occurrences were reported by Loeb 86 in 17 and 19 year old male siblings and by Wallman 175 in a father and daughter. In 5 of the 28 families described by Diamond et al., two children were involved. 28 Mott el al. 109 and Forare 40 have reported separate families in which multiply affected children had the same father but different mothers. It was suggested that in these families the condition was caused by a dominant gene with reduced expressivity in the paternal parent. The variable family patterns described do not present a clear picture of the inheritance of congenital hypoplastic anemia or, for that matter, whether heredity is a consistent factor. It is likely, in view of the rarity of this disorder, that until the basic defect is identified the true role of inheritance will remain unknown. Pathogenesis. Several hypotheses have been advanced regarding the etiology of congenital hypoplastic anemia. The occurrence of multiply affected individuals within families, the increased incidence of congenital malformations, and the presence of anemia at birth in some patients indicates that prenatal influences are involved. Unfortunately, detailed genetic studies are not available to clarify whether inherited or environmental factors are of major importance. Review of maternal drug histories indicates that only occasionally have mothers taken drugs during gestation which are known myelosuppressive agents. 28 , 67 Blood group isoimmunization does not appear to be a factor. 28 , 65 Chromosomal studies have been normal in all but one reported patient. 12, 166 Several authors have reported that patients with congenital hypoplastic anemia have an increased excretion of tryptophan metabolites in their urine, specifically anthranilic acid, O-aminohippuric acid, and kyn-

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urenine. 6 , 92,121 It was suggested that erythroid hypoplasia may be related to a metabolic abnormality in this pathway. However, Price et al. 128 recently studied tryptophan metabolism in 15 patients and only found slightly increased amounts of kynurenine and hydroxykynurenine in a few. No large quantities of free anthranilic acid or its conjugates were detected. It was reasoned that the occasional abnormalities found in some patients may be related to factors other than those specific for congenital hypoplastic anemia. Abnormal excretion of tryptophan metabolites have been reported in patients with various types of anemia, particularly those characterized by diminished production of erythrocytes. 56 Once the humoral factors governing normal human erythropoiesis were elucidated and a renal hormone with physiologically important erythropoietic stimulating characteristics identified, it was considered that congenital hypoplastic anemia may represent a primary deficiency of this homeostatic system. Hammond et al. studied erythropoietin levels in serum and urine of 22 patients with congenital hypoplastic anemia, however, and found elevated levels in all,54 A direct relationship existed between the severity of anemia and the level of erythropoietin activity. Seemingly, this discounted a role for erythropoietin. However, it was recognized that since erythropoietin determinations were done by a biological assay method, the possibility of an incomplete erythropoietin in affected patients supplemented by factors in the plasma of the test animals could not be excluded. Accordingly, the same group of investigators infused fresh normal plasma into 8 patients with congenital hypoplastic anemia and observed increased marrow erythroid activity and reticulocytosis in four. 55 The remaining patients failed to respond. It was suggested that a component of normal plasma required to activate, protect, or transport erythropoietin may be lacking in some patients with congenital hypoplastic anemia. An explanation for the etiology of congenital hypoplastic anemia may be sought in the erythropoietic alterations which occur physiologically after birth. The normal infant is born with a high reticulocyte count which rapidly decreases to low levels during the first week of life}47 Erythropoietin levels are elevated in cord blood and presumably in utero in association with the increased erythropoietic demands during this period. 53 The prompt subsidence of erythropoietic activity during the neonatal period is accompanied by the disappearance of detectable levels of erythropoietin in infant plasma during the early weeks of life. The demonstration of an inhibitor of erythropoiesis during the first week of life 156 suggests that a perturbation of these normal physiologic events with an increased level or premature appearance of an inhibitory substance may be of etiologic importance in congenital hypoplastic anemia. A number of clinical and laboratory observations indicate that multiple etiologies may be responsible for congenital hypoplastic anemia. These include variable responses to corticosteroid therapy, response of some but not all patients to infusions of fresh normal plasma, varied inheritance patterns, and a spectrum of bone marrow pictures from marked depletion of erythroid elements to various patterns of disordered erythropoiesis. 65 ,90 It seems reasonable to conclude that the major pathogenetic mechanism is a decrease in the differentiation and flow of stem cells

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through the erythron. This may be due to an inhibitory factor, a primary intracellular metabolic disturbance, or failure of adult erythropoietic regulatory mechanisms. Therapy. The course of congenital hypoplastic anemia has been significantly influenced by the observation of Gasser in 1951 that corticosteroids can induce bone marrow erythropoietic activity.44 The first extensive experience with steroids was reported by Allen and Diamond in a series of 22 patients. 3 Twelve of these children developed a reticulocytosis followed by normalization of hemoglobin, hematocrit, and erythrocyte counts. Subsequent studies have confirmed the effectiveness of corticosteroids in the treatment of congenital hypoplastic anemia;65, 67, 90,155 however, not all patients respond. Of the 6 cases reported by Sjolin and Wranne, 3 responded with a prompt reticulocytosis while 3 others did not improve. 155 Therapy with corticosteroids should be administered to all patients with congenital hypoplastic anemia in an initial dose of 20 to 30 milligrams of prednisone or its equivalent daily. Response is heralded by a brisk reticulocytosis within 4 to 11 days. Continued therapy with prednisone will be required in most patients to maintain satisfactory hemoglobin levels. Since permanent remission may not be achieved for years, the growth suppressing effects of corticosteroid therapy are of paramount importance and may lead to significant impairment in linear growth. 3 In most patients, this problem can be circumvented by maintaining the minimal dose of corticosteroids necessary to sustain hemoglobin levels of 10 to 12 gm. per 100 mI. and periodically attempting to lower the steroid dosage in small increments of 2.5 to 5 mg. daily at 4 to 6 week intervals. The long normal erythrocyte survival time and the sensitivity of the disease to small fluctuations in dosage levels dictates that greater change in medications may lead to erratic control. It has also been found that daily administration of corticosteroids is not necessary to maintain satisfactory hemoglobin levels and, in addition, may lead to more significant growth retardation than an intermittent program of therapy. Several regimens can be used in which corticosteroids are administered 3 or 4 days each week, daily during alternate weeks, or every other day of each week. 3 ,155 No superiority of anyone of these regimens has been demonstrated. The mechanism by which corticosteroids exert their effect in congenital hypoplastic anemia is unknown. Allen and Diamond have suggested that stimulation of a critical enzyme may be involved. 3 Cortisone has been shown to stimulate the liver enzyme tryptophan peroxidase;77 however, there is no indication that this enzyme is involved in the genesis of congenital hypoplastic anemia. Corticosteroids may exert their effect by interfering with anti-erythrocyte antibodies which have been shown to play a role in several adults with the acquired form of the disease. 70 , 79, 80.172 It seems likely that until the pathogenesis of congenital hypoplastic anemia is defined, the mechanism of steroid action will remain unknown. In patients who do not respond to corticosteroids, no other form of therapy has been of proven value. Periodic blood transfusions are necessary with their attendant risk of hemosiderosis. Most of these patients

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will show evidence of growth and sexual retardation, hypersplenism, and osteoporosis. 28 Despite these untoward developments, however, hope should not be abandoned. Several authors have observed the development of spontaneous remissions after years of multiple transfusions. 28 . 65 Careful records of pre and post transfusion hemoglobin levels as well as the amount and frequency of blood administered should be maintained so that the development of a spontaneous remission can be recognized promptly.

APLASTIC ANEMIA Aplastic anemia may be defined as a primary bone marrow disturbance characterized by diminished cellularity and pancytopenia without evidence of infiltrative disease adenopathy, or hepatosplenomegaly. The spectrum of aplastic anemia has been broadened by some authors to include those pancytopenias which are associated with hyperplasia as well as hypoplasia of bone marrow precursors. l07 In this context, aplastic anemia is viewed as a pancytopenia resulting from inadequate cell production regardless of the anatomic state of the bone marrow. 111 Despite the appeal of this functional approach to the concept of aplastic anemia, however, peripheral cytopenias associated with bone marrow hyperplasia have been reported predominantly in adults 26 so that application of this broad definition to children is less meaningful. Consequently, the more restricted definition will be retained in referring to pediatric patients in this review. Both acquired and congenital causes of aplastic anemia are known to occur. Since the clinical, laboratory, therapeutic, pathogenetic and prognostic considerations in each differ, they will be considered separately. Constitutional (Congenital) Aplastic Anemia The concept that aplastic anemia may occur on the basis of an inborn defect of bone marrow function was introduced by Fanconi in 1927. 37 He described three brothers, aged 5 to 7 years, who developed a fatal anemia. The constitutional nature of the disorder was suggested by its familial occurrence and the findings of microcephaly, hyperpigmentation, hypogenitalism, and hyperreflexia. Since this original report, approximately 160 cases of pancytopenia have been described in which evidence for a prenatal origin existed. The designation "Fanconi's anemia" was proposed for this disorder by Naegeli in 1931. 112 However, recently the term constitutional aplastic anemia has been used to refer to any syndrome of pancytopenia and bone marrow hypoplasia in which evidence of familial occurrence or congenital onset suggests an inherent defect.12. 57 In this context, Fanconi's aplastic anemia refers to a specific subgroup. Although it is recognized that opinions differ concerning the most suitable classification for these marrow aplasias,61. 161 the advent of recent diagnostic and investigative tools suggests that the syndrome is a heterogeneous one and that further subcategorization will likely evolve (Table 2). F ANCONI'S APLASTIC ANEMIA. Fanconi's anemia is the most frequently occurring of the constitutional aplastic anemias. It represents

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Classification of Constitutional Aplastic Anemias

I. Fanconi's panmyelopathy A. With congenital malformations B. Without congenital malformations II. Onset of amegakaryocytic thrombopenia in early infancy'''' III. Dyskeratosis congenita IV. Association with pancreatic insufficiency'''' and metaphyseal dysostosisI6 7 V. Association with immune deficiency disorders"

a pancytopenia caused by inadequate bone marrow production, which is associated with a multiplicity of congenital malformations or a family history of similarly involved siblings, and usually, characteristic cytogenetic abnormalities. Clinical. Many reports have described the plethora of congenital anomalies which occur in Fanconi's anemia. 2,24,34,96, lOS, 114, 117 Certain organ systems are preferentially involved; however, marked variation of malformations between patients and even among affected siblings occurs, The most common congenital defects involve the skeleton, skin, kidneys, and central nervous system (Table 3). Occasionally, malformations are minimal or absent and the syndrome is identified because of similarly affected siblingsY5. 115, 185 The onset of hematologic manifestations usually occurs between 4 and 7 years, although cases have been reported with symptoms originating in infancy through the second decade of life. 24 , 34, 71, 97, 132 Most series have reported an overall sex incidence of 3:2, with males predominating. 24 , 7\, 122 However, when analyzed according to familial and sporadic cases, male predominance is present only in those patients with a positive family history.117 The disease has been reported in many racial groups, although it is rare in Negroes. 126,171 In a few instances, the diagnosis has been suspected prior to the onset of hematologic abnormalities in patients with typical congenital abnormalities,71 a positive family history,t21 elevated fetal hemoglobin levels 148 or characteristic cytogenetic changes,61' 143, 171, 185 The usual presenting symptoms, however, are related to the gradual evolution of pancytopenia. In contrast to acquired bone-marrow aplasia, symptoms in Fanconi's anemia are more insidious and in many patients are present for a year or more before diagnosis. Laboratory Findings. The central feature of the peripheral blood in Fanconi's anemia is pancytopenia. During the early phases of the disease, elevated reticulocyte counts are often found (2 to 6 per cent) and, in some instances, marked increases up to 40 per cent have been noted. 164 With progression of the disease, reticulocytopenia dominates. Erythrocyte morphology is typically hyperchromic and macrocytic and prompted Fanconi in his preemptory report to propose the designation "perniciosiform anemia."37 Reports of bone marrow morphology and cellularity vary considerably. Usually the aspirate is hypocellular and shows fatty replacement. Scattered islands of hematopoietic tissue are found with relative predominance of erythroid precursors in varying stages of development. These cells tend to be large in size and show evidence of nuclear cytoplasmic dissociation. In addition, a variety of nuclear and mitotic abnormali-

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ties have been observed including polyploidy, anaphase bridges, multipolar divisions, and micronucleU 41 Elevated fetal hemoglobin values have been reported in most patients and in a few antedated the development of peripheral blood abnormalities. 148 A variety of metabolic functions have been studied in patients with Fanconi's anemia. Either normal or inconsistent abnormalities l2 , 126 have been found in folic acid and vitamin BI2 metabolism, urinary amino acid patterns, adrenal function, tryptophan metabolism, and plasma proteins. Autologous red cell survival studies have indicated that erythrocyte life span is normal in some patients 24 , 115, 126 and shortened in others.114, 122, 154.164 Ferrokinetic studies demonstrate low incorporation of radiolabeled iron into circulating erythrocytes associated with either rapid or slow disappearance from the plasma. 154 Numerous attempts have been made to

Table 3. Congenital Abnormalities Found in Fanconi's Aplastic Anemia Skeletal Absent, hypoplastic, or supernumerary thumbs Reduced ossification centers of the wrist Hypoplasia or absence of the radius Absent forearm Hypoplasic thenar eminence Syndactylism Sprengel's deformity Scoliosis, cervical rib, congenital hip dislocation, club foot Skin pigmentation Diffuse or mottled Cafe au lait spots Renal Aplasia Duplication of pelvis or ureters Horseshoe kidney Renal ectopy Central nervous system Microcephaly Mental retardation Microophthalmia Hyperreflexia Eye and ear anomalies Growth retardation Endocrine Pituitary insufficiency Hypogonadism Miscellaneous Cardiovascular disease Osteoporosis Obesity Absent radial pulses

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identify a consistent intrinsic metabolic abnormality in Fanconi's anemia erythrocytes because of the frequently elevated reticulocyte counts and decreased red cell survival. Both normal and abnormal values have been reported for erythrocyte glycolysis, hexokinase, glucose-6-phosphate dehydrogenase, ATP and ATP-ase. 49 • 143. 144. 164 The basis for these variable results is unknown but they may be related to different phases of disease in which patients were studied or to heterogeneity of the underlying defect. Cytogenetics. In 1964 Schroeder et al. described the presence of several unusual cytogenetic findings in two brothers with Fanconi's anemia. 142 In contrast to conventional chromosomal aberrations, 25 per cent of the cells from these two patients showed a variety of structural alterations which indicated an increased susceptibility of their chromosomes to undergo breakage. Subsequently, more than 40 patients with Fanconi's anemia have been described with similar cytogenetic abnormalities. In the two largest series reported, 29 to 35 total patients (82 per cent) had abnormal chromosomal findings. 12. 141 Similar observations have not been described in other types of constitutional aplastic anemia. Three types of chromosomal alterations have been found in Fanconi's anemia (Figure 1). The most frequent abnormality noted is chromatid breakage, usually affecting a single chromatid, although occasionally both chromatids at adjacent sites may be involved. The breakage is associated with rotation or angulation of the distal fragment

o Figure 1. Cytogenetic findings in Fanconi's anemia. A, Chromatid breakage; B, C, chromatid exchanges; D, endoreduplicated metaphase.

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and in some instances the chromosomal material distal to a break is missing. In general, there is no predilection for the breakage to involve any particular group or pair of chromosomes. The most characteristic of the cytogenetic abnormalities are chromatid exchanges. There are "star-like" recombination figures apparently arising as a result of chromatid breaks in two adjacent chromosomes followed by nonhomologous reunion of the chromatid ends. The least frequent abnormalities found are endoreduplications. In these figures, duplication of each chromosome occurs without separation of the partners so that the cell is composed of 46 chromosomal pairs. The incidence of metaphases with chromatid breaks varies from 6 to 75 per cent. Chromatid exchanges may be found in up to 25 per cent of mitotic figures but are not related to the frequency of chromatid breaks. Endoreduplicated figures are the least frequent of the cytogenetic changes in Fanconi's anemia and seldom represent more than 10 per cent of the cells examined. Chromosomal studies in family members of patients with Fanconi's anemia have not regularly shown an increased incidence of chromosomal breakage, chromatid exchanges, or endoreduplications. 12 . 122. 173.177 The chromosomal abnormalities are predominant in peripheral blood lymphocyte cultures stimulated by mitogen. In most instances, fibroblast cultures and direct preparations from dividing bone marrow cells do not reveal the abnormalities demonstrable in lymphocytes. 12 . 61. 143 When fibroblast and bone marrow studies have been abnormal, the frequency of aberrant cells was less than that found in concurrently studied peripheral blood lymphocyte preparations. 161 Malignant Transformation. An increased incidence of malignancy in Fanconi's anemia was first pointed out in 1959 by Garriga and Crosby.43 These authors reviewed 66 cases occurring in 48 families and determined that the incidence of leukemia in family members exceeded that expected in the normal population. Subsequently, the increased incidence of leukemia has also been noted in affected patients. In toto, 8 cases have been reported in Fanconi's anemia homozygotes. 31 The cell type involved has usually been of myeloid or monocytic origin in contrast to the more common lymphoblastic leukemia which occurs in normal children. In addition to the increased incidence of leukemia in Fanconi's anemia, other malignancies may also occur more frequently. Two patients with hepatic tumors 21.127 and four with carcinomas have been described.34. 97, 161, 162 An in vitro correlate of the increased tendency toward malignant transformation in Fanconi's anemia was demonstrated by Todaro et al. ,169 who developed a quantitative assay for studying the susceptibility of human diploid fibroblasts to transformation in culture by oncogenic viruses. Two homozygotes showed an 8- and 16-fold increase in transformation susceptibility compared to normal patients, whereas the heterozygotes showed intermediate values between normals and patients with the fully developed syndrome. More recently, Miller and Todaro indicate that the range of susceptibility to fibroblast transformation may be more nearly equal in Fanconi's anemia homozygotes and heterozygotes. 104

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The finding of increased transformability of fibroblasts in Fanconi's anemia and other congenital and acquired disorders carrying a high risk of cancer suggests that some individuals may have an increased susceptibility to malignancy because of an inherent genetic defect. 98 Since close, phenotypically normal relatives of patients with Fanconi's anemia have been shown to have abnormal fibroblast transformabilitY,61, 104, 169 Swift analyzed the cause of death in 106 near relatives of 8 probands. 163 A malignant neoplasm was the cause of death in 27 patients compared to an expected incidence of 17.4 in an equivalent normal population. In addition, among 372 living relatives, eight had a history of carcinoma or sarcoma. Although it was not possible to correlate the development of malignancy with fibroblast transformation in these patients, it could be estimated that the average risk of a Fanconi's anemia heterozygote dying from cancer was three times that of a noncarrier. Pathogenesis. Despite a swell of clinical, family, metabolic, and cytogenetic data in Fanconi's anemia, it must be concluded that the cause of the disease is still unknown. A number of factors have emerged, however, which may have important bearing on the etiology. The hereditary characteristics of Fanconi's anemia are well known. 33 , 117 The frequency of multiple cases in sibships, the high incidence of parental consanguinity, and the 25 per cent occurrence rate in affected sibs hips when corrected for ascertainment suggest an autosomal recessive mode of inheritance. This type of transmission has been questioned, however, because of the variability of findings among affected individuals within a given sibship,24, 96, 130 the occurrence of a few anomalies but not the entire expression of Fanconi's anemia among relatives of patients,24, 130 the rare instances of mother-child cases,68, 118 the increased average age of mothers at the time of birth of probands,36 the tendency for affected children to follow in order after the first patient in a sibship presents,36 the increased incidence in males, and the variability of erythrocyte metabolic studies, Although some of these conflicting data may have been introduced because of bias in case selection for reporting, collectively they indicate that a simple mode of inheritance cannot explain all of the findings in Fanconi's anemia and that heterogeneity or increased complexity of genetic factors probably exists, The cytogenetic aberrations which are present in most patients with Fanconi's anemia have raised speculation that they may be the cause of the syndrome, including the increased susceptibility to malignant transformation, However, it has not been possible to incriminate the cytogenetic abnormalities directly in the etiology of the congenital malformations and pancytopenia because of their nonspecific nature and occurrence in other disorders which differ phenotypically from Fanconi's anemia,12, 139 In addition, they are detected primarily in lymphocytes which have been artificially stimulated to undergo division and not consistently in direct bone marrow preparations or fibroblast cultures, so that their in vivo occurrence has not been established. Their absence in acquired forms of bone marrow aplasia,12, 22 however, documents a specificity for Fanconi's anemia and suggests they are at least casually related to the basic underlying alteration in this disorder, It has been hypothesized that they are caused by a deficiency of essential intracellular me-

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tabolites 141 or to increased breakdown by lysosomal enzymes;161 however, direct evidence for these hypotheses is lacking. Humoral substances in the plasma of patients causing increased breakage have not been found. 12. 49,122 Their major utility at present is a diagnostic one, since they are found in patients with both major and minor congenital malformations. They are of particular value in patients where the characteristics of the pancytopenia suggest Fanconi's anemia but only minor congenital malformations, such as slight hyperpigmentation or microophthalmia, render a clinical diagnosis difficult. 96 . 185 The concept that cytogenetic abnormalities in Fanconi's anemia may be the basis for malignant transformation is an intriguing one. The presence of chromosomal hyperfragmentation in other situations in man and animals which are known to be associated with oncogenicity, such as viral infections, irradiation exposure, and chemical contact, is consistent with the hypothesis. 12 Furthermore, in Bloom's syndrome 139 and other inherited disorders l43 both chromosomal breakage and a high incidence of leukemia have been described. The finding of increased chromosomal breakage in Fanconi's anemia lymphocytes following exposure to irradiation 60 and alkylating agents 144 further suggests the malignant potentiality of these cells. Nevertheless, it must be concluded that the evidence for chromosomal fragmentation as an etiologic factor in malignant transformation is indirect. Final hearing on this issue must await the demonstration of these cytogenetic changes in vivo and further study of other rare syndromes, such as dyskeratosis congenita which bears many similarities to Fanconi's anemia including pancytopenia, congenital malformations, and an increased susceptibility to malignant transformation!. 23. 42,78.157 but in which abnormal cytogenetic findings have not as yet been found.17.102 Prognosis. The long-range outlook for children with Fanconi's anemia cannot be accurately defined. Prior to the use of andro gen therapy, few patients lived more than 2 years following the onset of hematologic abnormalities. With hormone therapy, however, the majority of patients respond and enter a partial remission which may persist for years. A few may remit permanently;24, 61, 95,177 however, the possibility that a greater incidence of malignancy will be encountered as patients live longer must be considered.

Acquired Aplastic Anemia Etiology. Acquired bone marrow failure may occur following exposure to a variety of myelotoxic agents or spontaneously without apparent cause. Incrimination of a drug as the etiologic agent in aplastic anemia is usually circumstantial since no satisfactory laboratory test or animal model system exists for confirmation. Furthermore, the frequent administration of multiple drugs and the coexistence of other clinical conditions, particularly infections, further complicate the assignment of a causative factor. Several categories of myelotoxins require consideration in the etiology of aplastic anemia. (1) The antineoplastic drugs and irradiation produce bone marrow depression regularly in direct proportion to their dose and duration of exposure. (2) Another group of agents is considered potentially myelotoxic since aplastic anemia occurs infrequently

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and their importance as a cause of marrow aplasia has been established by the frequency with which affected patients have been exposed to them. The American Medical Association Registry of Blood Dyscrasia has maintained a cumulative tabulation of reports since 1957 and has established a listing of potentially myelotoxic compounds.1O, 110 These include chloramphenicol, benzene, potassium perchlorate, phenothiazines, sulfonamides, phenylbutazone, gamma benzene hexachloride, gold salts, methylphenylethyl hydantoin, and tolbutamide. A history of administration within 6 months of symptoms should be elicited for these agents to be considered of etiologic importance. (3) Other drug exposures are often revealed in the histories of patients with aplastic anemia; however, ubiquitous use and concurrent administration with established myelotoxins question their importance in the genesis of marrow suppression. 20 Included in this category are antihistamines, acetylsalicylic acid, penicillin, streptomycin, tetracycline, and chloral hydrate. (4) Bacterial infections represent a well documented but infrequent cause of aplastic anemia. 4s , 63 Recent interest has arisen concerning the relationship of viral hepatitis to bone marrow aplasia. In 1955 Lorenze and Quaisar reported the first case of aplastic anemia following infectious hepatitis,B7 Since then, more than 30 additional patients with this syndrome have been described. The majority have been males between the ages of2 and 20 years. The onset of pancytopenia may present from 2 to 24 weeks following the episode of hepatitis, although most appear within 2 to 9 weeks. No correlation with the severity of hepatitis or the morphologic appearance of the liver has emerged. The majority of patients die; nevertheless, the outcome is not invariably fatal, as 5 reported patients have recovered. 134 Although relatively few examples of this syndrome have been described, many others are likely to have escaped reporting. The possibility that some cases of "idiopathic" aplastic anemia may occur in patients who have had anicteric hepatitis requires consideration. One such patient has been reported. 134 Since viral illnesses occur frequently in children, it may be that other viral agents are important in patients without readily demonstrable causes for marrow aplasia. Although extensive virological studies have not been conducted in children with aplastic anemia, at least one other example of viral induced bone marrow asplasia with pancytopenia in man is known. 62 (5) Pancytopenia and bone marrow hypoplasia may be the presenting clinical expression of other hematologic diseases. Paroxysmal nocturnal hemoglobinuria,s4 a rare acquired erythrocyte disorder in childhood, and acute leukemia66 ,99 are occasionally signaled by a premonitory phase of marrow hypoplasia suggesting aplastic anemia. It is not clear whether marrow aplasia is an early manifestation of these disorders or the primary event which renders the bone marrow more susceptible to the other conditions. The relative incidence of the various causes of aplastic anemia in childhood is difficult to establish. In most early reports, children were included along with adults and a clear differentiation of the etiologies within each age category was not made. Recently, several series of aplastic anemia in children have been reported by O'Gorman Hughes,6:l Killander et al.,73 and Heyn et al. 59 A total of 143 cases were described by

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these authors. Fifty per cent were drug induced, 31 per cent were idiopathic, 6 per cent were due to chemicals or toxins, and 12 per cent represented miscellaneous causes. Upon comparing the studies separately, however, it becomes apparent that marked variability exists. For example, drug-induced causes represented 21, 45, and 82 per cent within the individual series, whereas patients in whom no demonstrable cause could be established accounted for 9, 32, and 63 per cent of the total. The basis for this variability is not clear; however, geographic and racial factors as well as the periods of time during which the cases were accumulated may in part account for the differences. Clinical Findings. The clinical manifestations of acquired aplastic anemia usually develop gradually over 2 to 6 weeks. If detected early, thrombocytopenia or thrombocytopenia and leukopenia may be the predominant features; however, depression of all three major cell types invariably follows. The anemia is characteristically normochromic and normocytic or moderately macrocytic. Reticulocytopenia, both relative and absolute, is a prominent early finding and represents direct evidence of bone marrow failure. The lowered white blood cell count is primarily due to depression of the granulocytic component with sparing of lymphoid elements. Thrombocytopenia is an invariable early feature of the disease and platelet counts less than 50,000 per cu. mm. are usually found. The bone marrow is mildly to severely hypocellular. In an occasional patient, the initial marrow aspirate may reveal normal or increased cellularity due to the presence of patchy areas of activity in an otherwise generally aplastic marrow. 124 Repeat aspirates or preferably biopsy by needle or surgical curettage will reveal the true marrow character. 83 • 111 Examination of the aspirate indicates a deficiency of all hematopoietic precursors with a relative increase in lymphocytes, mast cells, and histocytes. Megaloblastoid features of erythroid precursors characterized by increased size and abnormal nuclear clumping may be seen. Laboratory Studies. The serum iron and plasma iron binding capacityare elevated. Folic acid and vitamin B12 levels are usually normal. An increase in alkali resistant (fetal) hemoglobin is often present. 148 Ahaptoglobinemia64 and a positive acid hemolysis or sugar water test indicates the possibility of paroxysmal nocturnal hemoglobinuria. 84 The i antigen, a blood group ordinarily found only in cord blood and up to 18 months of age, is usually detectable on the erythrocytes of children with aplastic anemia regardless of ageY Other red cell investigations85 including intracellular enzymes, susceptibility to complement lysis, ultrastructural membrane abnormalities and ability to absorb antibody have not revealed consistent alterations. Erythrokinetic studies characteristically, but not invariably, demonstrate normal red cell survivaP7. 85. 131 Ferrokinetic investigations show a prolonged plasma radioiron disappearance curve with diminished appearance of label in circulating red cells. 46 • 94. 124. 131 Cytogenetic analyses reveal a normal diploid number of chromosomes without structural alterations. 12 • 22 Plasma and urinary erythropoietin levels are increased. 82 Pathogenesis. Relatively little is known concerning the mechanisms of bone marrow depression in aplastic anemia. The variable etiologic agents and clinical courses suggest that multiple primary defects

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may exist and that one or more may be operative in individual patients. Several pathogenetic concepts are suggested by currently existing data. 1. Predictable depression of hematopoiesis occurs following the use of irradiation and antineoplastic drugs by interference with the synthesis or replication of deoxyribonucleic acid. It is possible that other myelotoxic drugs which infrequently produce bone marrow aplasia may act similarly in susceptible individuals because of increased absorption, decreased excretion, or limited detoxification. 174 , 178, 183 Chloramphenicol, for example, has been shown to exert a direct, concentration related effect on bone marrow precursors as the likely explanation for one of its two recognized myelotoxicities. 186 All patients treated with chloramphenicol, if given sufficient amounts of the drug, will develop an erythropoietic lesion characterized by maturation arrest of bone marrow precursors, vacuolization of early erythroblasts and occasionally immature granulocytes, reticulocytopenia, increased plasma iron, decreased plasma clearance of radioiron, and occasionally anemia, leukopenia, or thrombocytopenia. The essential features of this form of toxicity are its uniform reversibility and close correlation with chloramphenicol blood levels. These observations suggested a direct metabolic effect of the drug on bone marrow precursors; however, initial efforts to demonstrate a biochemical defect in reticulocytes similar to the impaired amino acid incorporation in bacteria (antibiotic action) were unsuccessful. 183 Subsequently, chloramphenicol has been shown to significantly inhibit erythrocyte mitochondrial protein synthesis. Recent studies suggest that this effect is related to its myelotoxicity since both rabbit and human bone marrow mitochondrial protein synthesis is significantly altered by doses of chloramphenicol within the therapeutic range, whereas this effect is not produced by other non-myelosuppressive antibiotics. 91 The functional impairment is also accompanied by an ultrastructural modification of the mitochondrial matrix which is correlated with levels of free chloramphenicol. 182 This predictable effect of chloramphenicol upon bone marrow precursors is to be distinguished from the rare, late (onset usually longer than one month after the last dose of drug), non-dose related, often fatal bone marrow aplasia. A sequential relationship between the two types of chloramphenicol induced bone marrow suppression has not been established; however, in our present state of knowledge it seems unreasonable to continue the drug in the face of evidence indicating early hematopoietic suppression. 2. Evidence indicating an inherent or genetic basis for bone marrow aplasia has been established in constitutional aplastic anemia. The development of aplastic anemia in siblings without congenital malformations, either spontaneous 35 , 115, 185 or drug-induced,38.113 indicates that inborn errors may also predispose phenotypically normal individuals to the potential myelotoxic effects of certain agents. 13 . 113 Yunis et al. have demonstrated that patients who recovered from chloramphenicol induced aplastic anemia and their close relatives show an impaired in vitro uptake of 14C-Iabeled formate into DNA and RNA derived from bone marrow precursors exposed to the drug. 183 These observations suggest that an inherited biochemical predisposition in nucleic acid production, which is

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responsible for the development of aplastic anemia, is present in these patients. 3. The observation that an allergic history is often obtained in patients with aplastic anemia and the development of thrombocytopenic purpura and agranulocytosis following immune destruction l4 , 106 suggest a similar mechanism for bone marrow aplasia. Osgood 119 hypothesized that toxic agents may combine with specific cell proteins to form an antigenic complex against which immune antibodies are formed. These antibodies then react with and destroy the cell. Recently, further insight into a possible autoimmune basis for aplastic anemia has been gained from experimental investigations in rats which indicate that the bone marrow compartment is normally composed of two primary interrelated organs. 159 The hematopoietic tissue includes those cells undergoing continuous rapid proliferation and maturation in preparation for delivery to the peripheral blood. The matrix or superstructure of the marrow is composed of vascular and neural cells which replicate at a much slower rate. Transplantation studies following local and generalized bone marrow irradiation have demonstrated the essential requisite of this sinusoidal microcirculation for normal hematopoietic cell function. 76 In its absence even isogeneic cells will not survive. The potential importance of immune factors in the destruction of the sinusoidal system as a basis for marrow aplasia is revealed by the disruption and disappearance of this adventitial tissue in animals who develop marrow aplasia during the graft versus host syndrome following allogeneic marrow transplant and after the administration of bone marrow antibodies. 8 1, 116 The importance of the marrow microcirculation in some humans with aplastic anemia is suggested by the lack of uniform success with isogeneic marrow infusions obtained from identical twins 123 and the observation that marrow aplasia may be anatomically irregular with scattered areas of seemingly normal hematopoiesis in an otherwise generally hypoplastic marrow. These observations have been interpreted to indicate that destruction of the microcirculation rather than hematopoietic precursors may explain failures in isogeneic grafting or repopulation of aplastic areas of marrow by surrounding normal tissue. 75 Recent successes with bone marrow transplants from HL-A matched siblings to aplastic recipients, however, indicate that in many patients the microenvironment of the bone marrow is intact. 16S Successful restitution of marrow activity in these cases argues for a deficiency of hematopoietic stem cells as the primary defect. Prognosis. Many attempts have been made to correlate clinical and laboratory findings with the ultimate outcome of aplastic anemia; however, no satisfactory parameters have been established. 59, 73, 83 Age of onset, sex, etiology, severity of aplasia as measured by peripheral blood counts and bone marrow cellularity, quantitative fetal hemoglobin levels, and bone marrow lymphocyte counts have been analyzed and either conflicting or unconfirmed results reported. The primary issues are whether the nature of the inciting agent or the degree of marrow suppression is related to prognosis and whether specific hormone therapy alters the outcome of the disease. In the absence of reliable established quantitative indicators, prognosis is best judged by the overall severity of pancy-

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topenia and bone marrow depletion. Children with the more extreme cellular deficiencies are those in whom the outlook for recovery is poorest.8. 25. 73. 151 Reports prior to 1960 indicated that sustained clinical and hematologic remissions in aplastic anemia were rare. In 1957, Wolff reported a 3.3 per cent remission rate in 334 cases of whom 24.3 per cent were less than 15 years of age. 179 Shahidi and Diamond described a 5 per cent spontaneous remission rate among 40 children studied between 1938 and 1958. 149 Scott et al. 145 and Mohler and Leavell 107 reported no remissions in patients under 20 years of age. Since 1960, more favorable reports have appeared suggesting general improvement in the prognosis of aplastic anemia during childhood. However, the relative importance of hormone therapy and improved techniques of supportive care in effecting this change have not been clearly established because of the lack of controlled prospective studies. Shahidi and Diamond first reported the results of combined corticosteroid-androgen therapy in 17 cases of acquired aplastic anemia, 9 of whom responded to therapy and required no medication for 3 to 22 months. 151 Similar observations were recorded by Desposito et al. in 5 of 9 children with acquired aplastic anemia who showed sustained hematologic improvement on combined hormone therapy without the need for continuing medication. 25 In 1965, Lewis reviewed 60 patients studied over a 12 year period of whom 13 were less than 15 years of age. 8 :J Eighteen of the total were alive and six had sustained a complete remission, although it was not possible to determine whether corticosteroid or combined corticosteroid-androgen therapy was a factor in patients who did well. O'Gorman Hughes, in 1966, reviewed 104 cases of acquired aplastic anemia including some of those described earlier by Shahidi and Diamond 150 and observed a 34 per cent remission rate in patients treated with corticosteroids and androgens and 19 per cent in those managed symptomatically.64 Positive responses were seen by Allen et aU in 4 consecutive children treated with oxymethalone, a synthetic derivative of testosterone. Sanchez-Medal documented a 43 per cent remission rate among 14 patients less than 15 years of age who were treated with oxymethalone. 137 Killander reported in 1969 that of 19 children treated with a combination of corticosteroids and androgens 11 responded initially; however, in only three was it possible to discontinue therapy.73 Heyn et al. in the same year described 33 children with acquired aplastic anemia who were treated with supportive care alone. 59 Sixteen died; however, 17 recovered and only 2 required continued blood transfusions. Although the results are not uniform, studies within the past decade indicate substantial improvement in the overall gross reported prognosis of children with aplastic anemia in contrast to patients described in earlier series. It may be too early to conclude, however, that an improved prognosis can be anticipated for all patients with aplastic anemia. For example, 10 of 11 consecutive children with acquired marrow aplasia and pancytopenia seen at the University of Florida College of Medicine since 1961 have diedY The exceptional case is dependent on hormone therapy and may have a constitutional basis for her disease. The causes of aplasia

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in these patients were chloramphenicol in 4, hepatitis in 2 and no discernible cause in 5. Combined corticosteroid-androgen (testosterone, 6; oxymethalone, 4) therapy was administered to each patient in this group and no improvement was observed in the 9 who succumbed.

THERAPY The treatment of acquired and congenital aplastic anemia in childhood has been extensively reviewed recently by Diamond and Shahidp6 and Pochedly.125 These references should be consulted for details of supportive care, treatment of complications, and the rationale and precautions concerning the use of combined corticosteroid-androgen therapy. The upsurge of interest during the past 10 years in the concept of transplantation and the identification of major histocompatibility loci in man have offered additional avenues of potential therapy in aplastic anemia. It has been repeatedly observed that a major cause of death in aplastic anemia is hemorrhage, usually gastrointestinal or intracranial, secondary to thrombocytopenia. The only successful treatment for this major complication is platelet replacement. Although platelet concentrates or platelet rich plasma prepared from random donors is successful initially in preventing hemorrhage, continued administration leads to alloimmunization rendering further transfusions ineffective. Recently, it has been demonstrated that platelets obtained from histocompatible siblings and transfused to patients who demonstrate refractoriness to platelets from random donors survive normally. 52 Long-term administration of platelets from appropriately selected family members for periods up to 77 weeks has repeatedly provided excellent clinical responses and, when available, may be instrumental in prolonging life. 52 The necessity of using older siblings for donors, however, will limit the utility of this approach in families with only younger children. With the demonstration by Lorenz et al. in 1951 that bone marrow transplantation could prevent death in animals exposed to lethal doses of irradiation,88 the era of replacement therapy for bone marrow aplasia arrived. Initial efforts were discouraging, however, and a review of bone marrow transplants prior to 1969 indicated the futility of this approach in 73 patients with aplastic anemia. 15 No evidence of chimerism was demonstrated and studies failed to indicate that any of the patients benefited from this procedure. Enthusiasm waned until the advent of tissue typing techniques and a clearer understanding of the genetics of histocompatibility in man were developed. 74 Currently, a recrudescence of interest in bone marrow transplantation as a primary form of treatment for aplastic anemia has emerged l70 and initial results are promising. The utility of bone marrow transplantation has been explored in 3 groups of diseases-leukemia, aplastic anemia, and immune deficiency syndromes. The results with immunologic disorders have been particularly promising since several examples of chimerism and reconstitution of immune competency have been demonstrated. loo • 135, 15 Thomas et al. have reviewed the recent experience with bone marrow transplantation in aplastic anemia and reported on the use of this technique in 4 patients,

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aged 12 to 60 years, with severe bone marrow failure. 168 All 4 patients showed early evidence of marrow engraftment with an increase in marrow cellularity and peripheral blood counts. Two patients have maintained functioning grafts up to 7 months following the transplant. In retrospect, it is now clear that the basis for early failures with bone marrow transplantation was the lack of recognition that a major histocompatibility system exists in man as in animals. 32 This precludes the use of donors except for those siblings who can be demonstrated to carry the same transplantation antigens as the recipient. 138 Although proper donor selection by currently existing techniques does allow the successful engrafting of bone marrow, it does not insure uniform uncomplicated successes.100.1:18 The risks of graft versus host disease (GVHD) and graft rejection are still significant problems. Treatment of GVHD with immune suppressive agents and antilymphocyte serum, however, is sufficiently effective to minimize the danger of this potentially lethal complication. 93 Adequate preconditioning of the patient with cyclophosphamide has proven of value in diminishing the problem of graft rejection. 170 A further problem to be solved concerns the most suitable technique of processing marrow prior to infusion. Ideally, a fraction rich in stem cells and poor in immunocompetent cells would be most suitable. 7 Methods for preparing such fractions by gradient sedimentation techniques based on cell size or density are currently in the experimental stage 29 . 30. 103 and hopefully will expand the applicability and diminish the risk of bone marrow transplantation. The preliminary results with human bone marrow transplantation are likely to herald a new dimension in the treatment of aplastic anemia. The techniques of patient and donor selection, conditioning of the patient to minimize the risk of graft versus host disease and graft rejection, fractionation of the marrow to provide a stem cell rich fraction, and support of the patient during the critical post-transplant period are sufficiently advanced to indicate that their solution is technically feasible. A limiting factor at the present time for the pediatric patient is the requirement for a histocompatible sibling who is old enough to serve as a suitable donor.138

ACKNOWLEDGMENT

The author wishes to thank Dennis Short, Division of Cytogenetics, Department of Pathology for permission to reproduce the cytogenetic figures.

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5. Al-Mondhiry, H., Zanjani, E. D., Spivack, M., et al.: Pure red cell aplasia and thymoma: Loss of serum inhibitor of erythropoiesis following thymectomy. Blood, 38:576, 1971. 6. Altman, K. I., and Miller, G.: A disturbance of tryptophan metabolism in congenital hypoplastic anemia. Nature, 172:868, 1953. 7. Amato, D., Bergsagel, D. E., Clarysse, A. M., et al.: Review of bone marrow transplants at the Ontario Cancer Institute. Transplant Proc., 3:397, 1971. 8. Bernard, J., and Najean, Y.: Evolution and prognosis of the idiopathic pancytopenias. Series Haemat., 5:1, 1965. 9. Best, W. R.: Chloramphenicol-associated blood dyscrasias. A review of cases submitted to the American Medical Association Registry. J.A.M.A., 201 :181, 1967. 10. Best, W. R: Drug-associated blood dyscrasias. Recent additions to the registry. J.A.M.A., 185:286, 1963. 11. Bloom, G. E.: Unpublished observations. 12. Bloom, G. E., Warner, S., Gerald, P. S., and Diamond, L. K.: Chromosome abnormalities in constitutional aplastic anemia. New Eng. J. Med., 274:8, 1966. 13. Boga, M., and Szemere, P. A.: Infectious hepatitis and aplastic anemia-in two sisters. Lancet, 2:708, 1971. 14. Bolton, F. G.: Thrombocytopenic purpura due to quinidine. II. Serologic mechanisms. Blood, 11 :547,1956. 15. Bortin, M. M.: A compendium of reported human bone marrow transplants. Transplantation, 9:571, 1970. 16. Brittingham, T. E., Lutcher, C. L., and Murphy, D. L.: Reversible erythroid aplasia induced by diphenylhydantoin. Arch. Intern. Med., 113: 764, 1964. 17. Bryan, H. G., and Nixon, R K.: Dyskeratosis congenita and familial pancytopenia. J.A.M.A., 192:203, 1965. 18. Burgert, E. 0., Jr., Kennedy, R L. J., and Pease, G. L.: Congenital hypoplastic anemia. Pediatrics, 13:218, 1954. 19. Chernoff, A. J., and Josephson, A. M.: Acute erythroblastopenia in sickle cell anemia and infectious mononucleosis. Amer. J. Dis. Child., 82:310, 1951. 20. Clement, D. H.: Aplastic anemia. PEDIAT. CLIN. N. AMER., 9:703,1962. 21. Clinicopathologic Conference: Fanconi's anemia and hepatic cirrhosis. Amer. J. Med., 39:464, 1965. 22. Cobo, A., Lisker, R, Cordova, M. S., and Pizzuto, J.: Cytogenetic findings in acquired aplastic anemia. Acta Haemat., 44:32, 1970. 23. Cole, H. N., Cole, H. N., Jr., and Lascheid, W. P.: Dyskeratosis congenita. Arch. Derm., 76:712, 1957. 24. Dawson, J. P.: Congenital pancytopenia associated with multiple congenital anomalies (Fanconi type). Pediatrics, 15:325, 1955. 25. Desposito, F., Akatsuka, J., Thatcher, L. G., and Smith, N. J.: Bone marrow failure in pediatric patients. I. Cortisone and testosterone treatment. J. Pediat., 64:683, 1964. 26. Diamond, L. K., and Shahidi, N. T.: Treatment of aplastic anemia in children. Seminars Hemat., 4:278, 1967. 27. Diamond, L. K., and Blackfan, K. D.: Hypoplastic anemia. Amer. J. Dis. Child., 56:464, 1938. 28. Diamond, L. K., Allen, D. M., and Magill, F. B.: Congenital (erythroid) hypoplastic anemia. A 25-year study. Amer. J. Dis. Child., 102:403, 1961. 29. Dicke, K. A., and Van Bekkum, D. W.: Allogeneic bone marrow transplantation after elimination of immunocompetent cells by means of density gradient centrifugation. Transplant. Proc., 3:666, 1971. 30. Dicke, K. A., Van Hooft, J. I. M.,and Van Bekkum, D. W.: The selective elimination of immunologically competent cells from bone marrow and lymphatic cell mixtures. II. Mouse spleen cell fractionation on a discontinuous albumin gradient. Transplantation, 6:562, 1968. 31. Dosik, H., Hsu, L. Y., Todaro, G. J., et al.: Leukemia in Fanconi's anemia: Cytogenetic and tumor virus susceptibility studies. Blood, 36:341,1970. 32. Editorial: Bone-marrow transplantation: Unexpected results. Lancet, 1 :26, 1971. 33. Editorial: Grouchy, J. de: Genetic diseases, chromosome rearrangements and malignancy. Ann. Intern. Med., 65:603, 1966. 34. Esparza, A., and Thompson, W. R: Familial hypoplastic anemia with multiple congenital anomalies (Fanconi's syndrome)-Report of three cases. Rhode Island Med. J., 49: 103, 1966. 35. Estren, S., and Dameshek, W.: Familial hypoplastic anemia of childhood. Amer. J. Dis. Child., 73:671, 1947. 36. Fanconi, G.: Familial constitutional panmyelocytopathy, Fanconi's anemia (F. A.). I. Clinical aspects. Seminars Hemat., 4:233, 1967. 37. Fanconi, G.: Familiiire infantile periziosaartige aniiemie (permiziiises Blutbild und Konstitution). J. G. Kinderheilk., 117:257, 1927.

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38. Fernbach, D. J., and Trentin, J. J.: Isologous bone marrow transplantation in an identical twin with aplastic anemia. In Proceedings of the Eighth International Congress of Hematology, vol. 1. Tokyo, Pan-Pacific Press, 1960, pp. 150-155. 39. Finkel, H. E., Kimber, R J., and Dameshek, W.: Corticosteroid-responsive acquired pure red cell aplasis in adults. Amer. J. Med., 43:771, 1967. 40. Fiirare, S. -A.: Pure red cell anemia in step siblings. Acta Paediat. Scand., 52:159,1963. 41. Foy, H., Kondi, A., and Macdougall, L.: Pure red-cell aplasia in marasmus and kwashiorkor treated with riboflavine. Brit. Med. J., 1 :937, 1961. 42. Garb, J.: Dyskeratosis congenita with pigmentation, dystrophia unguium, and leukoplakia oris. Arch. Derm., 77:704, 1958. 43. Garriga, S., and Crosby, W. H.: The incidence of leukemia in families of patients with hypoplasia of the marrow. Blood, 14:1008, 1959. 44. Gasser, C.: Aplastische Anamie (chromische erythroblastophthise) und cortison. Schweiz Med. Wschr., 81: 1441, 1951. 45. Gasser, C.: Aplasia of erythropoiesis. PEDIAT. CLIN. N. AMER., 4:445,1957. 46. Gevirtz, N. R, and Berlin, N. I.: Erythrokinetic studies in severe bone marrow failure of diverse etiology. Blood, 18:637, 1961. 47. Giblett, E. R, and Crookston, M. C.: Agglutinability of red cells by anti-i in patients with thalassaemia major and other haematological disorders. Nature, 201: 1138, 1964. 48. Glasser, R M., Walker, R. I., and Herion, J. C.: The Significance of hematologic abnormalities in patients with tuberculosis. Arch. Intern. Med., 125:691, 1970. 49. Gmyreh, D., Witkowski, R, Syllm-Rapoport, I., and Jacobasch, G.: Chromosomal aberrations and abnormalities of red cell metabolism in a case of Fanconi's anemia before and after development of leukemia. German Med. Monthly, 13:105, 1968. 50. Goodman, S. B., and Block, M. H.: A case of red cell aplasia occurring as a result of anti tuberculosis therapy. Blood, 24:616, 1964. 51. Griggs, R C.: Lead poisoning: Hematologic aspects. Progr. Hemat., 4:117,1964. 52. Grumet, F. C., and Yankee, R. A.: Long-term platelet support of patients with aplastic anemia. Effect of splenectomy and steroid therapy. Ann. Intern. Med., 73: 1, 1970. 53. Halvorsen, S.: Plasma erythropoietin levels in cord blood and in blood during the first weeks of life. Acta Paediat. Scand., 52:425, 1963. 54. Hammond, D., and Keighley, G.: The erythrocyte-stimulating factor in serum and urine in congenital hypoplastiC anemia. Amer. J. Dis. Child., 100:466, 1962. 55. Hammond, D., Shore, N., and Movassaghi, N.: Production, utilization and excretion of erythropoietin: I. Chronic anemias. II. Aplastic crisis. III. Erythropoietic effects of normal plasma. Ann. N.Y. Acad. Sci., 149:516, 1968. 56. Hankes, L. V., Brown, R R., Schiffer, L., and Schmae1er, M.: Tryptophan metabolism in humans with various types of anemias. Blood, 32:649, 1968. 57. Harris, J. W., and Kellermeyer, R W.: The Red Cell. Cambridge, Massachusetts, Harvard University Press, 1970, pp. 727-749. 58. Hathaway, W. E., Brangle, R W., Nelson, T. L., and Roeckel, I. E.: Aplastic anemia and alymphocytosis in an infant with hypogammaglobulinemia: Graft-versus-host reaction? J. Pediat., 68:713, 1966. 59. Heyn, R M., Ertel, I. J., and Tubergen, D. G.: Course of acquired aplastic anemia in children treated with supportive care. J.A.M.A., 208:1372,1969. 60. Higurashi, M., and Conen, P. E.: In vitro chromosomal radiosensitivity in Fanconi's anemia. Blood, 38:336, 1971. 61. Hirschman, R J., Shulman, N. R, Abuelo, J. G., and Whang-Peng, J.: Chromosomal aberrations in two cases of inherited aplastic anemia with unusual clinical features. Ann. Intern. Med., 71:107, 1969. 62. Imerslund, 0.: Hypoplastik anemi med multiple misdanne1ser (Fanconi-anemi). Nord. Med., 50:1301,1953. 63. Howie, D. L., and Crosby, W. H.: Bone marrow panhypoplasia in humans experimentally induced by viral infection. Blood, 18 :800, 1961. 64. Hughes, D. W. O'G.: Aplastic anaemia in childhood: A reappraisal. I. Classification and assessment. Med. J. Aust., 1 :1059,1969. 65. Hughes, D. W. O'G.: The varied pattern of aplastic anaemia in childhood. Aust. Paediat. J., 2:228, 1966. 66. Hughes, D. W. O'G.: Hypoplastic anaemia in infancy and childhood: Erythroid hypoplasia. Arch. Dis. Child., 36:349, 1961. 67. Huguley, C. M., Jr., Lea, J. W., and Butts, J. A.: Adverse hematologic reactions to drugs. Progr. Hemat., 5:105,1966. 68. Ibrahim, J. M., Rawstron, J., and Booth, J.: A case of red cell aplasia in a negro child. Arch. Dis. Child., 41 :213, 1966. 69. Jepson, J. H., Gardner, F. H., Degnan, T., and Vas, M.: A gamma globulin inhibitor of erythropoiesis in erythroblastopenic plasma from patients with thymoma. Clin. Res., 16:536,1968.

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