Case 14-2006 — A 25-Year-Old Woman with Anemia and Iron Overload
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《新英格兰医药杂志》
Presentation of Case
A 25-year-old woman was seen in the hematology clinic at this hospital because of anemia and laboratory evidence of increased iron stores.
The patient had been admitted to another hospital one month earlier because of pain in the left lower abdominal quadrant that radiated to the back and was associated with headache and shortness of breath. Computed tomography (CT) of the abdomen and pelvis showed calculi in the left kidney and fat stranding along the left iliopsoas muscle. Laboratory studies revealed a normochromic, normocytic anemia; the serum iron level was 213 μg per deciliter (38.1 μmol per liter); total iron-binding capacity, 222 μg per deciliter (39.8 μmol per liter); transferrin saturation, 96 percent; and ferritin, 711 ng per milliliter. Because of her progressive pain and the concern of her clinicians about the possibility of an iron-overload syndrome, she was transferred to this hospital 10 days later. Repeated CT of the pelvis disclosed a thrombosed left ovarian vein. She had been taking ethinyl estradiol and norethindrone for oral contraception for four months, and her clinician had recently increased the dose of estrogen to control breakthrough vaginal bleeding. She had been smoking 5 to 10 cigarettes per day for several years.
Therapy with heparin was begun, which was followed by the addition of warfarin. A hypercoagulability evaluation — tests for resistance to activated protein C (factor V Leiden); measurement of the levels of protein C, protein S, antithrombin III, and homocysteine; and tests for the presence of antiphospholipid antibodies (anticardiolipin IgG and IgM and lupus anticoagulant) or the prothrombin G20210A mutation — did not disclose an increased tendency toward thrombosis. The results of routine chemistry tests and urinalysis showed no abnormalities; the results of other tests are shown in Table 1. Bone marrow biopsy and aspiration were performed on the fourth hospital day, and specimens were obtained. The results were pending at the time of discharge.
Table 1. Results of Laboratory Tests.
The patient was discharged on the fifth hospital day, with instructions to continue taking low-molecular-weight heparin and warfarin and to follow up with a hematologist. The oral contraceptive was discontinued, and she was advised to stop smoking.
When seen at the hematology clinic, the patient said that she had felt well since her discharge. She reported a weight gain of 4.5 to 6.8 kg during the six months before admission. She worked in an office; she had discontinued smoking, consumed alcohol rarely, and did not use illicit drugs. She was in a monogamous relationship.
An episode of pneumococcal meningitis and omphalitis had occurred when the patient was only two months of age, which had been treated with antibiotics. During that illness, ascites developed but quickly resolved. Two years later, gastrointestinal bleeding developed, and portal hypertension caused by portal-vein thrombosis was diagnosed. After several hospitalizations for bleeding that required transfusions of packed red cells, a splenorenal shunt with splenectomy was performed when the patient was five years of age. The bleeding resolved, and she had had no further episodes of bleeding or anemia.
The patient was of Italian descent. Her father and paternal aunt had both received a diagnosis of Diamond–Blackfan anemia. Her father had not required further treatment after receiving a single transfusion at birth; her aunt, a patient of Dr. Diamond, had required transfusions from birth for chronic anemia and died of encephalitis at eight years of age. One of the patient's three sisters had had anemia since she was three years of age; a diagnosis of Diamond–Blackfan anemia was made, and she had been treated with corticosteroids and transfusions of packed red cells from 4 to 13 years of age. The sister, 33 years of age at the time of the patient's evaluation, ultimately required iron-chelation therapy, but had had no treatment for anemia since she was 15 years of age. The other two sisters, 32 and 30 years of age, did not have anemia. The patient's mother had had anemia as a child but had not required transfusions; she was told at the time of her eldest daughter's evaluation that she herself had -thalassemia trait.
The patient was allergic to aspirin, gentamicin, and macrolide antibiotics. She was taking penicillin (500 mg daily) for postsplenectomy prophylaxis, low-molecular-weight heparin, warfarin, and subdermal progesterone. A physical examination revealed no abnormalities.
Additional diagnostic tests were performed.
Differential Diagnosis
Dr. Eyal C. Attar: This patient presented with several hematologic problems. The first was deep venous thrombosis involving the left ovarian vein. This vein does not commonly undergo thrombosis in the absence of recent pregnancy or other pelvic conditions such as surgery or cancer, and 80 to 90 percent of thromboses occur on the right side. Because her hypercoagulability evaluation was negative, the thrombosis in this patient probably resulted from increased hypercoagulability from cigarette smoking and the estrogen in the oral contraceptives she used, in combination with altered venous anatomy resulting from her previous portal hypertension and splenorenal shunt. Treatment in this case involves six months of anticoagulation therapy, smoking cessation, and substitution of the estrogen-containing contraceptive with one containing only progesterone.
The second hematologic condition in this patient was a normochromic, normocytic anemia, with a family history of both Diamond–Blackfan anemia and -thalassemia trait. Finally, the third condition was iron overload without a history of excessive blood transfusions.
Anemia can be due to the loss, destruction, or decreased production of red cells, in descending order of acuity (Table 2). The need to correct coagulation defects and pursue endoscopic or surgical intervention necessitates the prompt diagnosis of acute hemorrhage. Aside from revealing a decrease in the levels of hemoglobin and the hematocrit, laboratory evaluation is often not helpful in the setting of acute blood loss, since there is inadequate time for reticulocytosis and an increase in the red-cell distribution width to develop. In this patient, the lack of clinical evidence of hemoptysis, melena, hematochezia, or pain makes acute hemorrhage unlikely.
Table 2. Differential Diagnosis of Anemia.
The second broad category, red-cell destruction, is typically associated with increased levels of lactate dehydrogenase, reticulocytosis, altered red-cell morphology, and bilirubinemia. Causes include microangiopathic hemolytic anemias, such as thrombotic thrombocytopenic purpura, the hemolytic–uremic syndrome, and disseminated intravascular coagulation. Patients with heparin-induced thrombocytopenia can also present with anemia and schistocytosis, but the symptoms are accompanied by thrombocytopenia and a history of heparin exposure, which are not present in this case. Autoimmune hemolytic anemia or hemoglobinopathies, such as sickle cell anemia, can result in low levels of ongoing hemolysis punctuated by episodes of rapid hemolysis. Schistocytes, characteristic of microangiopathic hemolytic anemias, and spherocytes, characteristic of autoimmune hemolytic anemia, are absent on this patient's peripheral-blood smear. Her Coombs' test was negative, which argues against autoimmune hemolytic anemia. There are no sickle-shaped red cells on her peripheral smear, nor did she have a history of hemolytic crises to suggest sickle cell disease. In summary, the absence of reticulocytosis and characteristic alterations in red-cell morphology combined with only very slightly elevated levels of lactate dehydrogenase and normal bilirubin levels make destruction an unlikely explanation for anemia in this patient.
Defective erythropoiesis represents the third category of causes of anemia. Causes include deficiencies of iron, vitamin B12, or folate; anemia of chronic disease; infiltrative bone marrow cancers; viral infections, such as with parvovirus B19; hemoglobinopathies, such as thalassemia that impair hematopoiesis; clonal disorders of bone marrow, such as myelodysplastic syndromes; and disorders such as Diamond–Blackfan anemia. Laboratory testing ruled out iron deficiency or vitamin deficiencies. The peripheral-blood smear has no morphologic features, such as myelophthisic changes, macrocytosis, Pelger–Hu?t-like cells, and granulocyte dysplasia, that might suggest bone marrow infiltration or a myelodysplastic syndrome. Parvovirus infection was ruled out by laboratory analyses of antiparvovirus IgG and IgM levels, which indicate previous exposure but not active infection.
This patient had a family history of both thalassemia and Diamond–Blackfan anemia. Her peripheral-blood smear shows target erythrocytes, which are typical of -thalassemia, which she could have inherited from her mother. However, this patient has normocytic red cells, whereas microcytosis is characteristic of thalassemia. Could she have inherited Diamond–Blackfan anemia from her father?1 More than 90 percent of cases of Diamond–Blackfan anemia are diagnosed within the first year of life, but the disease is sometimes detected later in childhood and occasionally in adulthood. It is typically characterized by a normochromic, macrocytic anemia, although neutropenia or thrombocytopenia may occur. Reticulocytes are rare or absent, as they were in this patient.
An evaluation of the patient's peripheral-blood smear must take into consideration the fact that she was asplenic. Asplenia can result in target red cells, Howell–Jolly bodies (nuclear remnants), Cabot rings, and Pappenheimer bodies (iron particles). However, asplenia would not result in anemia and iron overload. To help determine whether impaired hematopoiesis accounted for her anemia, a bone marrow aspiration and biopsy were performed.
Pathological Discussion
Dr. Robert P. Hasserjian: The bone marrow–biopsy specimen was moderately hypercellular for the patient's age, with a mild relative erythroid hyperplasia. The numbers of both early erythroid and myeloid forms were increased (Figure 1). The bone marrow–aspirate smear also showed preserved erythropoiesis with complete maturation and an increase in the numbers of early forms (proerythroblasts made up 11 percent of the erythroid cells, as compared with a mean of 3 percent in normal controls).2 Prominent dyserythropoiesis was present, including nuclear irregularities, nuclear fragmentation, and occasional binucleation (Figure 1B). An iron stain showed the presence of normal iron stores (3+ of 6+) and no pathologic ringed sideroblasts. Immunophenotyping by flow cytometry revealed no abnormal population of either B cells or T cells and no increase in the numbers of B-cell precursors (hematogones) or myeloid blasts. Cytogenetic analysis revealed a normal karyotype.
Figure 1. Specimens Obtained at Bone Marrow Biopsy and Aspiration.
The bone marrow specimen obtained by trephine biopsy (Panel A, Giemsa stain) is hypercellular, with a relative preponderance of erythroid elements, including increased numbers of early forms. The bone marrow aspirate (Panel B, Wright–Giemsa stain) contains maturing erythroid and myeloid elements, including frequent dysplastic late erythroid forms with nuclear fragmentation (arrow), as well as increased numbers of early erythroid forms (inset).
The interpretation of pathological specimens of bone marrow is greatly enhanced by knowledge of the clinical setting, such as the presence of an inherited or acquired condition that may affect bone marrow function. In this case, the question of Diamond–Blackfan anemia was raised because of the patient's family history and the low reticulocyte count. Patients with Diamond–Blackfan anemia typically have a paucity of erythroid elements in bone marrow owing to a proliferation defect of the erythroid lineage; the few erythroid elements present are usually pronormoblasts.3,4,5,6 In this patient, erythroid elements were normal in number and exhibited complete maturation. The findings of a hypercellular marrow with preserved erythropoiesis in the setting of reticulocytopenia suggested an erythroid maturation defect with ineffective erythropoiesis.
Congenital defects of DNA or hemogloblin synthesis may cause ineffective hematopoiesis. In such conditions, although erythropoiesis is preserved or even increased, the red-cell precursors undergo intramedullary cell death, resulting in a paucity of mature reticulocytes entering the circulation. Unfortunately, pathological findings in bone marrow alone are often not enough for the clinician to distinguish between the various causes of ineffective hematopoiesis. Although morphologic dysplasia is often associated with myeloid neoplasia and myelodysplastic syndromes, reactive morphologic dysplasia of the erythroid series may also occur as a result of infections, metabolic deficiencies, toxic effects, and hemoglobinopathies.7,8 Some authors have reported erythroid lineage dysplasia in cases of Diamond–Blackfan anemia,9,10 but dysplasia was not a prominent feature in one large series.11 In the series in which dysplasia was not a prominent feature, only 12 percent of the patients had normal or increased bone marrow erythropoiesis and reticulocytopenia, suggesting an arrest of maturation at the late normoblast stage, later than in patients with typical Diamond–Blackfan anemia; thus, the relative erythroid hyperplasia in this case does not preclude Diamond–Blackfan anemia contributing to this patient's anemia.
Differential Diagnosis
Dr. Attar: The bone marrow examination ruled out an infiltrative or neoplastic bone marrow disorder, and thus we turned our attention toward the causes of ineffective erythropoiesis.
Diamond–Blackfan Anemia
Diamond–Blackfan anemia is usually sporadic; familial cases are typically inherited in an autosomal dominant pattern, as in this family. Although a specific molecular defect has not been isolated, approximately 25 percent of patients have a mutation in the small ribosomal protein gene RPS19 located on chromosome 19q.12,13 A second gene, located on chromosome 8p, may account for approximately 40 percent of cases.14 Although the molecular basis of the disease is not known, the function of erythroid progenitor cells is compromised, as demonstrated by subnormal burst-forming unit–erythroid and colony-forming unit–erythroid in vitro activity, and progenitor cells from patients with this disease have reduced sensitivity to exogenous erythropoietin. Erythrocytes from patients with Diamond–Blackfan anemia contain elevated levels of fetal hemoglobin and erythrocyte adenosine deaminase, an enzyme of the fetal purine-salvage pathway.
Phenotypic heterogeneity is common in Diamond–Blackfan anemia. Even within families, the degree of anemia, response to treatment, and presence of congenital anomalies can vary.15 In about two thirds of patients, the anemia responds to treatment with exogenous glucocorticoids. Once a patient enters puberty, the anemia may resolve, as it did in this patient's sister, and it is postulated that the increase in production of endogenous steroids may obviate the need for supplementation. For patients with corticosteroid-refractory disease or for those who cannot tolerate corticosteroids, stem-cell transplantation from HLA-matched related donors has been performed.16
Although the bone marrow findings in this patient are not typical of Diamond–Blackfan anemia, this patient is an adult with only mild hematologic abnormalities, and thus, even if she has the disease, one would not expect to see the severe abnormalities associated with symptomatic infants. To assess whether the patient could have Diamond–Blackfan anemia, levels of erythrocyte adenosine deaminase were assessed in the patient and compared with those in other family members (Figure 2). She had an elevated level, similar to that of her father and her affected sister, suggesting that she inherited this disorder from her father.
Figure 2. Hematologic Values in the Patient and Her Family.
Solid symbols indicate family members who required blood products in childhood, suggesting the presence of Diamond–Blackfan anemia. One of the patient's sisters was not evaluated. eADA denotes erythrocyte adenosine deaminase, WC white cells, HgB hemoglobin, HCT hematocrit, PC platelet count, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, RTC reticulocyte count, HbA hemoglobin A, HbA2 hemoglobin A2, HbF hemoglobin F, and EU enzyme units. Data on eADA courtesy of Dr. Colin Sieff, Harvard Medical School, Children's Hospital, Boston.
-Thalassemia
Ineffective erythropoiesis can result from mutations that alter the quantity or function of hemoglobin, and because the patient's mother had been told she had -thalassemia, investigation of the status of the hemoglobin genes in this family is appropriate. Most persons carry two -globin genes on chromosome 16 and two -globin genes, a single -globin gene, and a single gene on chromosome 11 (Figure 3A). Other genes, including the -globin and -globin genes, contribute primarily to embryonic hemoglobin. Mutations that result in quantitative reductions in hemoglobin expression are termed "thalassemias," whereas those that alter hemoglobin function are termed "hemoglobinopathies." -thalassemia results from reduced expression of -globin and is associated with anemia, microcytosis, target cells, basophilic stippling, and an increase in hemoglobin A2, which is a result of increased binding of -globin to -globin.
Figure 3. Orientation of Embryonic, Fetal, and Adult Globin Genes and Mutations Present in This Family.
Panel A indicates the orientations of embyronic ( and ), fetal (), and adult ( and ) globin genes. The expression of globin genes proceeds from the 5' to the 3' direction through ontogeny under the control of regions located upstream of the globin gene clusters: hypersensitivity region 40 (HS40) for -globin and -globin and locus-control region (LCR) for -globin, -globin, -globin, and -globin. Hemoglobin molecules are made up of four globin chains. Embryonic hemoglobins are produced first, but hemoglobin F (22) becomes the dominant fetal hemoglobin at two months' gestation. Hemoglobin F binds oxygen more tightly than adult hemoglobin (HbA, 22), enhancing delivery of oxygen from maternal oxyhemoglobin to fetal deoxyhemoglobin at the placenta. In the postnatal period, -globin synthesis decreases and -globin and -globin genes contribute to the adult hemoglobins HbA (22) and HbA2 (22), which comprise approximately 97.5 percent and 2.5 percent of adult hemoglobin, respectively. The presence of a CT polymorphism in the G promoter (Panel B) and deletion of both -globin and -globin genes in this family contribute to increased hemoglobin F expression and decreased expression of hemoglobin A and hemoglobin A2. denotes pseudogene.
To assess whether this patient could have a thalassemia, hemoglobin electrophoresis was performed (Table 3). Whereas persons with -thalassemia have increased levels of hemoglobin A2 (5 to 7 percent) and decreased levels of hemoglobin A (93 to 95 percent), this patient had a hemoglobin A2 level of 1.8 percent, a hemoglobin A level of 70.6 percent, and a hemoglobin F level of 27.6 percent. To determine whether an elevated hemoglobin F level was an inherited characteristic or a condition that arose spontaneously in this patient, hemoglobin electrophoresis was performed on blood samples obtained from close family members (Figure 2). Both the patient's mother and her eldest sister had elevated levels of hemoglobin F, suggesting that this trait was inherited from the patient's mother. In contrast, however, both her mother and her sister had microcytic red cells.
Table 3. Results of Hemoglobin Electrophoresis in This Patient.
Elevated levels of hemoglobin F are observed in hereditary or acquired conditions that result in elevated levels of fetal hemoglobin, but they can also result from intentional pharmacologic manipulation.17,18 The similar findings on hemoglobin electrophoresis of the patient's blood and her mother's blood suggest a diagnosis of hereditary persistence of fetal hemoglobin, of which there are two types. The first type involves large deletions in both the -globin and -globin genes that result in increased binding of -globin to -globin.19 The second type involves nondeletional mutations in the promoter regions of -globin genes that result in increased transcription of -globin. One mutation, a CT transversion at base pair –158 in the G promoter that creates a site cleavable by the XmnI restriction enzyme,20 results in increased levels of hemoglobin F at times of erythrocyte stress, such as pregnancy.21 Other mutations involving the action of transcription regulators that are at a distance from the -globin genes are possible and still undergoing characterization.
We asked Dr. David Chui, professor of medicine at Boston University School of Medicine and director of the hemoglobin diagnostic reference laboratory at Boston Medical Center, for assistance in determining the molecular basis of this family's elevated levels of hemoglobin F. The patient's mother's blood was selected for further analysis, in order to eliminate the influence of Diamond–Blackfan anemia from the patient's father's side. Sequence analysis revealed four intact, nonmutated -globin genes. However, a 13.4-kb deletion involving the -globin and -globin genes, characteristic of Sicilian -thalassemia, was detected (Figure 3B). In addition, the mother also had an XmnI nondeletional mutation in the G promoter.
Persons with Sicilian -thalassemia have elevated hemoglobin F (6.0 to 19.0 percent in one series) and microcytosis, suggesting that this mutation did not fully account for the normocytosis and elevated levels of hemoglobin F in the case under discussion.19 The XmnI mutation is known to increase the production of -globin and result in hemoglobin F levels ranging from 2.3 to 3.8 percent in heterozygotes. The combined effect of these mutations is not completely understood. An analysis of 19 persons with Sicilian -thalassemia in an area with a high prevalence of the disorder in regions surrounding the Arachthos River in the Epirus region of Greece22 found that 10 persons who did not have the XmnI mutation had levels of hemoglobin F between 6.8 and 12.5 percent, 7 who were heterozygous for XmnI had hemoglobin F levels ranging from 7.3 to 9.8 percent, and 2 who were homozygous for XmnI had hemoglobin F levels of 8.1 and 9.7 percent. These values are still lower than those we observed in this patient.
These genetic studies indicate that both deletional and nondeletional mutations were present in this patient's family and therefore may account for her elevated levels of hemoglobin F and anemia, although other as yet unidentified mutations may increase hemoglobin F to levels exceeding those previously reported. It is probable that the presence of Diamond–Blackfan anemia in this patient has resulted in the absence of the typical microcytosis seen in -thalassemia, although this effect was not seen in the patient's similarly affected sister.
Iron Overload
Do these hemoglobin studies explain the patient's elevated iron levels and transferrin saturation? Although patients with severe thalassemia have iron overload from repeated blood transfusions, increased iron stores in patients with subclinical thalassemia are due to inappropriately increased gastrointestinal absorption. Athough the pathophysiological process is still under investigation, one explanation involves the molecule hepcidin, a small peptide that controls iron absorption from the gastrointestinal tract. Hepcidin, which increases in proportion to total body iron stores and limits gastrointestinal iron absorption, is inappropriately low in patients with thalassemia.23,24 Because iron overload is the main cause of complications and death in patients with thalassemia, further assessments of iron stores in the tissue of this patient were conducted to determine whether she should consider iron-chelation therapy. Her iron level was only mildly elevated at 4495 μg per gram of liver, dry weight, and we are not proceeding with iron chelation at the present time.
Dr. Morton N. Swartz (Infectious Diseases): Do you have any information on the next generation?
Dr. Attar: Neither the patient nor any of her siblings has children. However, genetic counseling and genotyping of future partners, particularly if any of the partners is of Mediterranean heritage, will be crucial to reduce the risk of thalassemia in their offspring.
A Physician: What is the relationship between the hemoglobinopathy and the hypercoagulability in this patient?
Dr. Attar: Patients with severe forms of -thalassemia have an increased risk of cerebral, portal-vein, and deep venous thrombosis in addition to pulmonary embolism.25 The causes are multifactorial. However, this patient's condition is similar to that seen with the -thalassemia trait, which is not associated with an increased clotting tendency. Instead, her risk factors for thrombosis include tobacco and estrogen use in combination with altered venous anatomy resulting from her venous shunting procedure.
Dr. Nancy Lee Harris (Pathology): Is there a mechanism to explain the response to corticosteroid treatment in Diamond–Blackfan anemia?
Dr. Attar: Ebert et al. have addressed this question.26 Targeted reduction of the RPS19 transcript in human CD34+ cells with the use of short hairpin RNA resulted in impaired proliferation and differentiation of erythroid progenitor cells that could be reversed with dexamethasone. Dexamethasone did not appear to alter transcription of RPS19 or other ribosomal genes, but rather it decreased the expression of genes specific to nonerythroid hematopoietic differentiation while increasing the expression of genes found in immature erythroid cells.
Anatomical Diagnoses
-thalassemia and XmnI mutation in the G hemoglobin promoter resulting in hereditary persistence of fetal hemoglobin.
Diamond–Blackfan anemia phenotype manifesting in elevated levels of erythrocyte adenosine deaminase and, possibly, macrocytosis, anemia, and impaired reticulocyte response.
No potential conflict of interest relevant to this article was reported.
We are indebted to Drs. David Nathan, Colin Sieff, Stuart Orkin, David Chui, and Wendy Garrett for their valuable input.
Source Information
From the Center for Leukemia (E.C.A.) and the Department of Pathology (R.P.H.), Massachusetts General Hospital; and the Departments of Medicine (E.C.A.) and Pathology (R.P.H.), Harvard Medical School — both in Boston.
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A 25-year-old woman was seen in the hematology clinic at this hospital because of anemia and laboratory evidence of increased iron stores.
The patient had been admitted to another hospital one month earlier because of pain in the left lower abdominal quadrant that radiated to the back and was associated with headache and shortness of breath. Computed tomography (CT) of the abdomen and pelvis showed calculi in the left kidney and fat stranding along the left iliopsoas muscle. Laboratory studies revealed a normochromic, normocytic anemia; the serum iron level was 213 μg per deciliter (38.1 μmol per liter); total iron-binding capacity, 222 μg per deciliter (39.8 μmol per liter); transferrin saturation, 96 percent; and ferritin, 711 ng per milliliter. Because of her progressive pain and the concern of her clinicians about the possibility of an iron-overload syndrome, she was transferred to this hospital 10 days later. Repeated CT of the pelvis disclosed a thrombosed left ovarian vein. She had been taking ethinyl estradiol and norethindrone for oral contraception for four months, and her clinician had recently increased the dose of estrogen to control breakthrough vaginal bleeding. She had been smoking 5 to 10 cigarettes per day for several years.
Therapy with heparin was begun, which was followed by the addition of warfarin. A hypercoagulability evaluation — tests for resistance to activated protein C (factor V Leiden); measurement of the levels of protein C, protein S, antithrombin III, and homocysteine; and tests for the presence of antiphospholipid antibodies (anticardiolipin IgG and IgM and lupus anticoagulant) or the prothrombin G20210A mutation — did not disclose an increased tendency toward thrombosis. The results of routine chemistry tests and urinalysis showed no abnormalities; the results of other tests are shown in Table 1. Bone marrow biopsy and aspiration were performed on the fourth hospital day, and specimens were obtained. The results were pending at the time of discharge.
Table 1. Results of Laboratory Tests.
The patient was discharged on the fifth hospital day, with instructions to continue taking low-molecular-weight heparin and warfarin and to follow up with a hematologist. The oral contraceptive was discontinued, and she was advised to stop smoking.
When seen at the hematology clinic, the patient said that she had felt well since her discharge. She reported a weight gain of 4.5 to 6.8 kg during the six months before admission. She worked in an office; she had discontinued smoking, consumed alcohol rarely, and did not use illicit drugs. She was in a monogamous relationship.
An episode of pneumococcal meningitis and omphalitis had occurred when the patient was only two months of age, which had been treated with antibiotics. During that illness, ascites developed but quickly resolved. Two years later, gastrointestinal bleeding developed, and portal hypertension caused by portal-vein thrombosis was diagnosed. After several hospitalizations for bleeding that required transfusions of packed red cells, a splenorenal shunt with splenectomy was performed when the patient was five years of age. The bleeding resolved, and she had had no further episodes of bleeding or anemia.
The patient was of Italian descent. Her father and paternal aunt had both received a diagnosis of Diamond–Blackfan anemia. Her father had not required further treatment after receiving a single transfusion at birth; her aunt, a patient of Dr. Diamond, had required transfusions from birth for chronic anemia and died of encephalitis at eight years of age. One of the patient's three sisters had had anemia since she was three years of age; a diagnosis of Diamond–Blackfan anemia was made, and she had been treated with corticosteroids and transfusions of packed red cells from 4 to 13 years of age. The sister, 33 years of age at the time of the patient's evaluation, ultimately required iron-chelation therapy, but had had no treatment for anemia since she was 15 years of age. The other two sisters, 32 and 30 years of age, did not have anemia. The patient's mother had had anemia as a child but had not required transfusions; she was told at the time of her eldest daughter's evaluation that she herself had -thalassemia trait.
The patient was allergic to aspirin, gentamicin, and macrolide antibiotics. She was taking penicillin (500 mg daily) for postsplenectomy prophylaxis, low-molecular-weight heparin, warfarin, and subdermal progesterone. A physical examination revealed no abnormalities.
Additional diagnostic tests were performed.
Differential Diagnosis
Dr. Eyal C. Attar: This patient presented with several hematologic problems. The first was deep venous thrombosis involving the left ovarian vein. This vein does not commonly undergo thrombosis in the absence of recent pregnancy or other pelvic conditions such as surgery or cancer, and 80 to 90 percent of thromboses occur on the right side. Because her hypercoagulability evaluation was negative, the thrombosis in this patient probably resulted from increased hypercoagulability from cigarette smoking and the estrogen in the oral contraceptives she used, in combination with altered venous anatomy resulting from her previous portal hypertension and splenorenal shunt. Treatment in this case involves six months of anticoagulation therapy, smoking cessation, and substitution of the estrogen-containing contraceptive with one containing only progesterone.
The second hematologic condition in this patient was a normochromic, normocytic anemia, with a family history of both Diamond–Blackfan anemia and -thalassemia trait. Finally, the third condition was iron overload without a history of excessive blood transfusions.
Anemia can be due to the loss, destruction, or decreased production of red cells, in descending order of acuity (Table 2). The need to correct coagulation defects and pursue endoscopic or surgical intervention necessitates the prompt diagnosis of acute hemorrhage. Aside from revealing a decrease in the levels of hemoglobin and the hematocrit, laboratory evaluation is often not helpful in the setting of acute blood loss, since there is inadequate time for reticulocytosis and an increase in the red-cell distribution width to develop. In this patient, the lack of clinical evidence of hemoptysis, melena, hematochezia, or pain makes acute hemorrhage unlikely.
Table 2. Differential Diagnosis of Anemia.
The second broad category, red-cell destruction, is typically associated with increased levels of lactate dehydrogenase, reticulocytosis, altered red-cell morphology, and bilirubinemia. Causes include microangiopathic hemolytic anemias, such as thrombotic thrombocytopenic purpura, the hemolytic–uremic syndrome, and disseminated intravascular coagulation. Patients with heparin-induced thrombocytopenia can also present with anemia and schistocytosis, but the symptoms are accompanied by thrombocytopenia and a history of heparin exposure, which are not present in this case. Autoimmune hemolytic anemia or hemoglobinopathies, such as sickle cell anemia, can result in low levels of ongoing hemolysis punctuated by episodes of rapid hemolysis. Schistocytes, characteristic of microangiopathic hemolytic anemias, and spherocytes, characteristic of autoimmune hemolytic anemia, are absent on this patient's peripheral-blood smear. Her Coombs' test was negative, which argues against autoimmune hemolytic anemia. There are no sickle-shaped red cells on her peripheral smear, nor did she have a history of hemolytic crises to suggest sickle cell disease. In summary, the absence of reticulocytosis and characteristic alterations in red-cell morphology combined with only very slightly elevated levels of lactate dehydrogenase and normal bilirubin levels make destruction an unlikely explanation for anemia in this patient.
Defective erythropoiesis represents the third category of causes of anemia. Causes include deficiencies of iron, vitamin B12, or folate; anemia of chronic disease; infiltrative bone marrow cancers; viral infections, such as with parvovirus B19; hemoglobinopathies, such as thalassemia that impair hematopoiesis; clonal disorders of bone marrow, such as myelodysplastic syndromes; and disorders such as Diamond–Blackfan anemia. Laboratory testing ruled out iron deficiency or vitamin deficiencies. The peripheral-blood smear has no morphologic features, such as myelophthisic changes, macrocytosis, Pelger–Hu?t-like cells, and granulocyte dysplasia, that might suggest bone marrow infiltration or a myelodysplastic syndrome. Parvovirus infection was ruled out by laboratory analyses of antiparvovirus IgG and IgM levels, which indicate previous exposure but not active infection.
This patient had a family history of both thalassemia and Diamond–Blackfan anemia. Her peripheral-blood smear shows target erythrocytes, which are typical of -thalassemia, which she could have inherited from her mother. However, this patient has normocytic red cells, whereas microcytosis is characteristic of thalassemia. Could she have inherited Diamond–Blackfan anemia from her father?1 More than 90 percent of cases of Diamond–Blackfan anemia are diagnosed within the first year of life, but the disease is sometimes detected later in childhood and occasionally in adulthood. It is typically characterized by a normochromic, macrocytic anemia, although neutropenia or thrombocytopenia may occur. Reticulocytes are rare or absent, as they were in this patient.
An evaluation of the patient's peripheral-blood smear must take into consideration the fact that she was asplenic. Asplenia can result in target red cells, Howell–Jolly bodies (nuclear remnants), Cabot rings, and Pappenheimer bodies (iron particles). However, asplenia would not result in anemia and iron overload. To help determine whether impaired hematopoiesis accounted for her anemia, a bone marrow aspiration and biopsy were performed.
Pathological Discussion
Dr. Robert P. Hasserjian: The bone marrow–biopsy specimen was moderately hypercellular for the patient's age, with a mild relative erythroid hyperplasia. The numbers of both early erythroid and myeloid forms were increased (Figure 1). The bone marrow–aspirate smear also showed preserved erythropoiesis with complete maturation and an increase in the numbers of early forms (proerythroblasts made up 11 percent of the erythroid cells, as compared with a mean of 3 percent in normal controls).2 Prominent dyserythropoiesis was present, including nuclear irregularities, nuclear fragmentation, and occasional binucleation (Figure 1B). An iron stain showed the presence of normal iron stores (3+ of 6+) and no pathologic ringed sideroblasts. Immunophenotyping by flow cytometry revealed no abnormal population of either B cells or T cells and no increase in the numbers of B-cell precursors (hematogones) or myeloid blasts. Cytogenetic analysis revealed a normal karyotype.
Figure 1. Specimens Obtained at Bone Marrow Biopsy and Aspiration.
The bone marrow specimen obtained by trephine biopsy (Panel A, Giemsa stain) is hypercellular, with a relative preponderance of erythroid elements, including increased numbers of early forms. The bone marrow aspirate (Panel B, Wright–Giemsa stain) contains maturing erythroid and myeloid elements, including frequent dysplastic late erythroid forms with nuclear fragmentation (arrow), as well as increased numbers of early erythroid forms (inset).
The interpretation of pathological specimens of bone marrow is greatly enhanced by knowledge of the clinical setting, such as the presence of an inherited or acquired condition that may affect bone marrow function. In this case, the question of Diamond–Blackfan anemia was raised because of the patient's family history and the low reticulocyte count. Patients with Diamond–Blackfan anemia typically have a paucity of erythroid elements in bone marrow owing to a proliferation defect of the erythroid lineage; the few erythroid elements present are usually pronormoblasts.3,4,5,6 In this patient, erythroid elements were normal in number and exhibited complete maturation. The findings of a hypercellular marrow with preserved erythropoiesis in the setting of reticulocytopenia suggested an erythroid maturation defect with ineffective erythropoiesis.
Congenital defects of DNA or hemogloblin synthesis may cause ineffective hematopoiesis. In such conditions, although erythropoiesis is preserved or even increased, the red-cell precursors undergo intramedullary cell death, resulting in a paucity of mature reticulocytes entering the circulation. Unfortunately, pathological findings in bone marrow alone are often not enough for the clinician to distinguish between the various causes of ineffective hematopoiesis. Although morphologic dysplasia is often associated with myeloid neoplasia and myelodysplastic syndromes, reactive morphologic dysplasia of the erythroid series may also occur as a result of infections, metabolic deficiencies, toxic effects, and hemoglobinopathies.7,8 Some authors have reported erythroid lineage dysplasia in cases of Diamond–Blackfan anemia,9,10 but dysplasia was not a prominent feature in one large series.11 In the series in which dysplasia was not a prominent feature, only 12 percent of the patients had normal or increased bone marrow erythropoiesis and reticulocytopenia, suggesting an arrest of maturation at the late normoblast stage, later than in patients with typical Diamond–Blackfan anemia; thus, the relative erythroid hyperplasia in this case does not preclude Diamond–Blackfan anemia contributing to this patient's anemia.
Differential Diagnosis
Dr. Attar: The bone marrow examination ruled out an infiltrative or neoplastic bone marrow disorder, and thus we turned our attention toward the causes of ineffective erythropoiesis.
Diamond–Blackfan Anemia
Diamond–Blackfan anemia is usually sporadic; familial cases are typically inherited in an autosomal dominant pattern, as in this family. Although a specific molecular defect has not been isolated, approximately 25 percent of patients have a mutation in the small ribosomal protein gene RPS19 located on chromosome 19q.12,13 A second gene, located on chromosome 8p, may account for approximately 40 percent of cases.14 Although the molecular basis of the disease is not known, the function of erythroid progenitor cells is compromised, as demonstrated by subnormal burst-forming unit–erythroid and colony-forming unit–erythroid in vitro activity, and progenitor cells from patients with this disease have reduced sensitivity to exogenous erythropoietin. Erythrocytes from patients with Diamond–Blackfan anemia contain elevated levels of fetal hemoglobin and erythrocyte adenosine deaminase, an enzyme of the fetal purine-salvage pathway.
Phenotypic heterogeneity is common in Diamond–Blackfan anemia. Even within families, the degree of anemia, response to treatment, and presence of congenital anomalies can vary.15 In about two thirds of patients, the anemia responds to treatment with exogenous glucocorticoids. Once a patient enters puberty, the anemia may resolve, as it did in this patient's sister, and it is postulated that the increase in production of endogenous steroids may obviate the need for supplementation. For patients with corticosteroid-refractory disease or for those who cannot tolerate corticosteroids, stem-cell transplantation from HLA-matched related donors has been performed.16
Although the bone marrow findings in this patient are not typical of Diamond–Blackfan anemia, this patient is an adult with only mild hematologic abnormalities, and thus, even if she has the disease, one would not expect to see the severe abnormalities associated with symptomatic infants. To assess whether the patient could have Diamond–Blackfan anemia, levels of erythrocyte adenosine deaminase were assessed in the patient and compared with those in other family members (Figure 2). She had an elevated level, similar to that of her father and her affected sister, suggesting that she inherited this disorder from her father.
Figure 2. Hematologic Values in the Patient and Her Family.
Solid symbols indicate family members who required blood products in childhood, suggesting the presence of Diamond–Blackfan anemia. One of the patient's sisters was not evaluated. eADA denotes erythrocyte adenosine deaminase, WC white cells, HgB hemoglobin, HCT hematocrit, PC platelet count, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, RTC reticulocyte count, HbA hemoglobin A, HbA2 hemoglobin A2, HbF hemoglobin F, and EU enzyme units. Data on eADA courtesy of Dr. Colin Sieff, Harvard Medical School, Children's Hospital, Boston.
-Thalassemia
Ineffective erythropoiesis can result from mutations that alter the quantity or function of hemoglobin, and because the patient's mother had been told she had -thalassemia, investigation of the status of the hemoglobin genes in this family is appropriate. Most persons carry two -globin genes on chromosome 16 and two -globin genes, a single -globin gene, and a single gene on chromosome 11 (Figure 3A). Other genes, including the -globin and -globin genes, contribute primarily to embryonic hemoglobin. Mutations that result in quantitative reductions in hemoglobin expression are termed "thalassemias," whereas those that alter hemoglobin function are termed "hemoglobinopathies." -thalassemia results from reduced expression of -globin and is associated with anemia, microcytosis, target cells, basophilic stippling, and an increase in hemoglobin A2, which is a result of increased binding of -globin to -globin.
Figure 3. Orientation of Embryonic, Fetal, and Adult Globin Genes and Mutations Present in This Family.
Panel A indicates the orientations of embyronic ( and ), fetal (), and adult ( and ) globin genes. The expression of globin genes proceeds from the 5' to the 3' direction through ontogeny under the control of regions located upstream of the globin gene clusters: hypersensitivity region 40 (HS40) for -globin and -globin and locus-control region (LCR) for -globin, -globin, -globin, and -globin. Hemoglobin molecules are made up of four globin chains. Embryonic hemoglobins are produced first, but hemoglobin F (22) becomes the dominant fetal hemoglobin at two months' gestation. Hemoglobin F binds oxygen more tightly than adult hemoglobin (HbA, 22), enhancing delivery of oxygen from maternal oxyhemoglobin to fetal deoxyhemoglobin at the placenta. In the postnatal period, -globin synthesis decreases and -globin and -globin genes contribute to the adult hemoglobins HbA (22) and HbA2 (22), which comprise approximately 97.5 percent and 2.5 percent of adult hemoglobin, respectively. The presence of a CT polymorphism in the G promoter (Panel B) and deletion of both -globin and -globin genes in this family contribute to increased hemoglobin F expression and decreased expression of hemoglobin A and hemoglobin A2. denotes pseudogene.
To assess whether this patient could have a thalassemia, hemoglobin electrophoresis was performed (Table 3). Whereas persons with -thalassemia have increased levels of hemoglobin A2 (5 to 7 percent) and decreased levels of hemoglobin A (93 to 95 percent), this patient had a hemoglobin A2 level of 1.8 percent, a hemoglobin A level of 70.6 percent, and a hemoglobin F level of 27.6 percent. To determine whether an elevated hemoglobin F level was an inherited characteristic or a condition that arose spontaneously in this patient, hemoglobin electrophoresis was performed on blood samples obtained from close family members (Figure 2). Both the patient's mother and her eldest sister had elevated levels of hemoglobin F, suggesting that this trait was inherited from the patient's mother. In contrast, however, both her mother and her sister had microcytic red cells.
Table 3. Results of Hemoglobin Electrophoresis in This Patient.
Elevated levels of hemoglobin F are observed in hereditary or acquired conditions that result in elevated levels of fetal hemoglobin, but they can also result from intentional pharmacologic manipulation.17,18 The similar findings on hemoglobin electrophoresis of the patient's blood and her mother's blood suggest a diagnosis of hereditary persistence of fetal hemoglobin, of which there are two types. The first type involves large deletions in both the -globin and -globin genes that result in increased binding of -globin to -globin.19 The second type involves nondeletional mutations in the promoter regions of -globin genes that result in increased transcription of -globin. One mutation, a CT transversion at base pair –158 in the G promoter that creates a site cleavable by the XmnI restriction enzyme,20 results in increased levels of hemoglobin F at times of erythrocyte stress, such as pregnancy.21 Other mutations involving the action of transcription regulators that are at a distance from the -globin genes are possible and still undergoing characterization.
We asked Dr. David Chui, professor of medicine at Boston University School of Medicine and director of the hemoglobin diagnostic reference laboratory at Boston Medical Center, for assistance in determining the molecular basis of this family's elevated levels of hemoglobin F. The patient's mother's blood was selected for further analysis, in order to eliminate the influence of Diamond–Blackfan anemia from the patient's father's side. Sequence analysis revealed four intact, nonmutated -globin genes. However, a 13.4-kb deletion involving the -globin and -globin genes, characteristic of Sicilian -thalassemia, was detected (Figure 3B). In addition, the mother also had an XmnI nondeletional mutation in the G promoter.
Persons with Sicilian -thalassemia have elevated hemoglobin F (6.0 to 19.0 percent in one series) and microcytosis, suggesting that this mutation did not fully account for the normocytosis and elevated levels of hemoglobin F in the case under discussion.19 The XmnI mutation is known to increase the production of -globin and result in hemoglobin F levels ranging from 2.3 to 3.8 percent in heterozygotes. The combined effect of these mutations is not completely understood. An analysis of 19 persons with Sicilian -thalassemia in an area with a high prevalence of the disorder in regions surrounding the Arachthos River in the Epirus region of Greece22 found that 10 persons who did not have the XmnI mutation had levels of hemoglobin F between 6.8 and 12.5 percent, 7 who were heterozygous for XmnI had hemoglobin F levels ranging from 7.3 to 9.8 percent, and 2 who were homozygous for XmnI had hemoglobin F levels of 8.1 and 9.7 percent. These values are still lower than those we observed in this patient.
These genetic studies indicate that both deletional and nondeletional mutations were present in this patient's family and therefore may account for her elevated levels of hemoglobin F and anemia, although other as yet unidentified mutations may increase hemoglobin F to levels exceeding those previously reported. It is probable that the presence of Diamond–Blackfan anemia in this patient has resulted in the absence of the typical microcytosis seen in -thalassemia, although this effect was not seen in the patient's similarly affected sister.
Iron Overload
Do these hemoglobin studies explain the patient's elevated iron levels and transferrin saturation? Although patients with severe thalassemia have iron overload from repeated blood transfusions, increased iron stores in patients with subclinical thalassemia are due to inappropriately increased gastrointestinal absorption. Athough the pathophysiological process is still under investigation, one explanation involves the molecule hepcidin, a small peptide that controls iron absorption from the gastrointestinal tract. Hepcidin, which increases in proportion to total body iron stores and limits gastrointestinal iron absorption, is inappropriately low in patients with thalassemia.23,24 Because iron overload is the main cause of complications and death in patients with thalassemia, further assessments of iron stores in the tissue of this patient were conducted to determine whether she should consider iron-chelation therapy. Her iron level was only mildly elevated at 4495 μg per gram of liver, dry weight, and we are not proceeding with iron chelation at the present time.
Dr. Morton N. Swartz (Infectious Diseases): Do you have any information on the next generation?
Dr. Attar: Neither the patient nor any of her siblings has children. However, genetic counseling and genotyping of future partners, particularly if any of the partners is of Mediterranean heritage, will be crucial to reduce the risk of thalassemia in their offspring.
A Physician: What is the relationship between the hemoglobinopathy and the hypercoagulability in this patient?
Dr. Attar: Patients with severe forms of -thalassemia have an increased risk of cerebral, portal-vein, and deep venous thrombosis in addition to pulmonary embolism.25 The causes are multifactorial. However, this patient's condition is similar to that seen with the -thalassemia trait, which is not associated with an increased clotting tendency. Instead, her risk factors for thrombosis include tobacco and estrogen use in combination with altered venous anatomy resulting from her venous shunting procedure.
Dr. Nancy Lee Harris (Pathology): Is there a mechanism to explain the response to corticosteroid treatment in Diamond–Blackfan anemia?
Dr. Attar: Ebert et al. have addressed this question.26 Targeted reduction of the RPS19 transcript in human CD34+ cells with the use of short hairpin RNA resulted in impaired proliferation and differentiation of erythroid progenitor cells that could be reversed with dexamethasone. Dexamethasone did not appear to alter transcription of RPS19 or other ribosomal genes, but rather it decreased the expression of genes specific to nonerythroid hematopoietic differentiation while increasing the expression of genes found in immature erythroid cells.
Anatomical Diagnoses
-thalassemia and XmnI mutation in the G hemoglobin promoter resulting in hereditary persistence of fetal hemoglobin.
Diamond–Blackfan anemia phenotype manifesting in elevated levels of erythrocyte adenosine deaminase and, possibly, macrocytosis, anemia, and impaired reticulocyte response.
No potential conflict of interest relevant to this article was reported.
We are indebted to Drs. David Nathan, Colin Sieff, Stuart Orkin, David Chui, and Wendy Garrett for their valuable input.
Source Information
From the Center for Leukemia (E.C.A.) and the Department of Pathology (R.P.H.), Massachusetts General Hospital; and the Departments of Medicine (E.C.A.) and Pathology (R.P.H.), Harvard Medical School — both in Boston.
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