Case Studies. A One-Year-Old Male Child With Progressive Pallor

Transcription

1 A One-Year-Old Male Child With Progressive Pallor Arun Gupta, MD, DNB, Geeta Gupta, MD, Taghreed Al Eisa, MBBS, Amani Al Shallal, MBBS, Hanan Al Wazzan, MRCPath, Rajaa Marouf, FRCPath (Department of Hematology, Mubarak Al-Kabeer Hospital, Kuwait University, Kuwait) DOI: /FV16VJ396VR5R6C7 Clinical History Patient One-year-old male Bedouin child. Chief Complaints/History of Present Illness Progressive pallor and poor weight gain of 3 months duration. Past Medical History This child was delivered at full term but was small for gestational age (birth weight 5.5 lbs). The antenatal period was unremarkable. The neonatal period was uncomplicated except for mild hyperbilirubinemia, which resolved with phototherapy. He was breast-fed for 6 months. Commercially available formula feeds were added 3 months prior when his mother noticed pallor. Family History He is the second child born of consanguineous marriage; the first child is a 2.6-year-old healthy female who has never been investigated for anemia. There is no history of hereditary disease. The family members have never been diagnosed with any hematological disorders. Physical Examination Vital signs: temperature 98ºF; pulse 96/min; blood pressure 90/60 mm Hg; weight 14 lbs 12 oz.; height 57 cm. The child was alert but irritable. Clinically, he appeared anemic with mild jaundice. Liver and spleen were palpable, each 1 cm below the costal margins. No lympadenopathy or any other physical abnormality was noted. Principal Laboratory Findings Table 1 Results of Additional Diagnostic Procedures Ultrasound of abdomen confirmed hepatosplenomegaly. No gallbladder stones were present. Hemoglobin electrophoresis was normal. A bone marrow aspirate revealed hypercellular marrow. Erythroid hyperplasia was noted with erythropoietic-to-granulopoietic ratio of 3. Marked dyserythropoiesis was observed predominantly in late erythroblasts (Image 2). Approximately 20% of erythroblasts were binucleated or trinucleated, and internuclear chromatin bridges were noted in 3% of them (Image 3). No ring sideroblasts were seen on Prussian blue staining. Analysis of red-cell membrane proteins by polyacrylamide gel electrophoresis with sodium dodecylsulphate (SDS-PAGE) showed a narrower and fastermoving band 3 (Image 4). Questions 1. What are this patient s most striking clinical and laboratory findings? 2. How do you explain the patient s most striking clinical and laboratory findings? 3. What conditions are suggested based on this patient s clinical and laboratory observations, and how should a patient with such findings be further investigated? 4. What is the most likely diagnosis? 5. What is the etiopathogenesis and molecular basis for this condition? 6. What options are available to treat patients with this disease? 7. What are the functional consequences and prognosis for patients with this disease? Possible Answers 1. The most striking clinical and laboratory observations are poor weight gain, progressive pallor, mild hepatosplenomegaly, low hemoglobin, high red-cell-distribution width, normal reticulocyte count, indirect hyperbilirubinemia, mildly reduced haptoglobin, and elevated lactate dehydrogenase levels. In addition, the peripheral blood smear shows anisopoikilocytosis and presence of basophilic stippling (Image 1). The serum acid lysis test (Ham test) is positive, but is negative when performed with autologous sera. The bone marrow aspirate shows erythroid hyperplasia and marked dyserythropoiesis (Image 2). Many erythroblasts show double- or triple-nuclei and internuclear chromatin bridges (Image 3). Furthermore, analysis of red cell membrane proteins by polyacrylamide gel electrophoresis with sodium dodecylsulphate (SDS-PAGE) shows a narrower and fastermoving band 3 (Image 4). 2. The patient s clinical and laboratory findings suggest anemia as a cause of his poor weight gain and pallor. He has mild splenomegaly, which is likely to contribute to anemia. The presence of anisopoikilocytosis on blood smear suggests disordered erythropoiesis caused by membrane defect and indicates damage to cells after their formation. Basophilic stippling is indicative of disturbed erythropoiesis and defect in maturation. Inadequate reticulocyte response to degree of anemia in peripheral blood suggests ineffective erythropoiesis. All these findings, together with erythroid hyperplasia and dyserythropoiesis in bone marrow, suggest anemia due to ineffective erythropoiesis. This is supported by the presence of Downloaded labmedicine.com from July 2008 j Volume 39 Number 7 j LABMEDICINE 405

2 Table 1_Principal Laboratory Findings Test Patient s Normal Result Reference Range Hematology White blood cell count x 10 3 /µl Red blood cell count x 10 6 /µl Hemoglobin g/dl Hematocrit 24 31% 41% MCV fl MCH pg MCHC g/dl RDW 26.5 <17% Platelet count x 10 3 /µl Reticulocyte count x 10 3 /µl Peripheral blood smear findings Image 1 Coagulation Prothrombin time sec Partial thromboplastin time sec Fibrinogen mg/dl D-dimer mg/dl Chemistry Total bilirubin mg/dl Indirect bilirubin mg/dl AST U/L ALT U/L LDH U/L Serum iron ug/dl Serum ferritin ng/ml Serum transferrin saturation 45% 20% 50% Serum B pg/ml Serum folate ng/ml RBC folate ng/ml Special tests Direct antiglobulin test Negative Negative G6PD assay IU/Hb Heat instability test Negative Negative Autohemolysis test Negative Negative Ham test Positive Negative Ham test with homologous sera Negative Negative Sucrose lysis test Negative Negative MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; RBC, red blood cell; Hb, hemoglobin; G6PD, glucose-6-phosphate dehydrogenase. Image 1_Patient s peripheral blood smear findings, illustrating anisopoikilocytosis and basophilic stippling. elevated serum LDH and indirect hyperbilirubinemia. A positive Ham test indicates a red cell membrane defect. Furthermore, a narrower and faster moving band 3 on SDS-PAGE confirms abnormality of red cell membrane. 3. The patient had evidence of ineffective erythopoiesis in the bone marrow, which is associated with anemia, jaundice, and hepatosplenomegaly. The conditions suggested are both congenital and acquired in nature. Particular attention should be given to investigating thalassemia, hereditary spherocytosis, autoimmune hemolytic anemia, red cell enzyme deficiencies (particularly glucose-6-phosphate dehydrogenase and pyruvate kinase deficiencies), megaloblastic anemia, and disorders of unstable hemoglobin. These should be evaluated by hemoglobin electrophoresis, direct Coombs test, glucose-6-phosphate dehydrogenase and pyruvate kinase assays, osmotic fragility test, heat instability, and autohemolysis tests. Serum B 12, folic acid, and red cell folate levels should be estimated to exclude megaloblastic anemia. If none of these indicate a definite diagnosis, a bone marrow examination should be performed to exclude congenital sideroblastic and congenital dyserythropoietic anemias (CDAs). Prussian blue staining should be done on bone marrow aspirate smears to exclude sideroblastic anemia. Examination of bone marrow aspirate for characteristic abnormalities is required for the diagnosis of CDA. If there is suggestion of CDA, a Ham test should be performed to identify CDA type II. A Ham test with autologous sera and sucrose lysis tests is required to differentiate paroxysmal nocturnal hemoglobinuria from CDA. Further analysis of red cell membranes by electron microscopy or SDS-PAGE is helpful in confirming red cell membrane abnormality. 4. Most likely diagnosis: Congenital dyserythropoietic anemia type II. Congenital dyserythropoietic anemias are a group of hereditary disorders of erythropoiesis, characterized by ineffective erythropoiesis, presence of multinuclearity in bone marrow erythroid precursors, and secondary hemosiderosis. 1 The clinical presentation of CDA is quite heterogeneous. The diagnosis is often made during childhood, though there is no age limit. It can have intrauterine onset with severe anemia or the disease may remain undiscovered until late adult life. 2 Many cases remain underdiagnosed as the clinical features of CDA are not pathognomonic and extensive investigations are required to exclude other causes of dyserythropoietic anemias. However, the diagnosis is suggested when there is evidence of anemia, jaundice, features of ineffective erythropoiesis, and a characteristic morphological appearance of bone marrow erythroblasts. 3,4 While 3 types of CDAs have been described (CDA I, CDA II, and CDA III), there are cases which fulfill the general definition of CDAs but do not fit into 1 of the 3 categories. These are classified as variants of CDA and have unknown genetic basis. 5 Congenital dyserythropoietic anemia I is characterized by congenital anemia, ineffective erythropoiesis, and characteristic morphological abnormalities on both light and electron microscopy. 6 Most patients are diagnosed during childhood or adolescence or later in life. 7 A few case reports have also suggested severe intrauterine anemia. 8 The anemia is macrocytic with a low reticulocyte count. In peripheral blood, large poikilocytes and elliptocytes are noted. Basophilic stippling is always present. 9 Nonhematological dysmorphologies, namely syndactylism of the hands and feet, Downloaded 406 from LABMEDICINE j Volume Number 7 j July 2008 labmedicine.com

3 Case Studies A B Image 2_Wright-Giemsa stain of patient s bone marrow aspirate illustrating (A) dyserythropoiesis and (B) a binucleate erythroblast. absence of nails, additional toes, and dyskeratosis-like skin pigmentation, are noted in approximately 50% of patients.10 Microscopic examination of the bone marrow shows a megaloblastoid aberration of chromatin structure, large polyploidal cells with an irregularly shaped nuclear mass often consisting of 2 segments and double- or triple-nucleated erythroblasts, and internuclear chromatin bridges. Thirty percent to A C Downloaded labmedicine.com from 60% of all erythroblasts are considered abnormal.11 Examination under an electron microscope shows clumps of heterochromatin in erythroblasts, giving the appearance of Swiss cheese. 12 Analysis of erythrocyte-membrane proteins by SDS-PAGE reveals below-normal levels of protein 4.5. Changes in red cell morphology and a decrease in protein 4.5 are highly distinct features of CDA I.12 B Image 3_Wright Giemsa stain of patient s bone marrow aspirate illustrating (A) many bi- and trinucleated erythroblasts and (B and C) internuclear chromatin bridges. July 2008 j Volume 39 Number 7 j LABMEDICINE 407

4 to any of the 3 types of CDA. This may be due to any other defined congenital anemia or as a result of an incomplete diagnostic workup. 16 This category includes CDA with prominent erythroblastosis that resembles CDA type II but is negative for acidified serum test, CDA with thrombocytopenia, CDA with intra erythroblastic inclusions, etc. 17 Image 4_SDS-PAGE of patient s red cell membrane illustrating a thin and faster-moving band 3. Congenital dyserythropoietic anemia II is the most frequently seen type of CDA. The principal manifestations include hepatosplenomegaly, variable normocytic normochromic anemia with indirect hyperbilirubinemia, inadequate reticulocytosis, and low or absent haptoglobin. 13 Associated physical abnormalities are less common compared with CDA I. 10 Red cell morphology is always abnormal and shows anisopoikilocytosis and basophilic stippling. Microscopic examination of bone marrow shows dyserythropoiesis, binuclearity or multinuclearity in 10% to 50% of mature erythroblasts, and internuclear chromatin bridges. 9 Electron microscopic examination shows elongated vesicles lining the inner surface of the plasma membrane of erythroblasts corresponding to endoplasmic reticulum remnants, giving the appearance of a double membrane. 12 In addition, the red cells of these patients display enhanced agglutination with anti-i antibodies and are lysed when incubated in acidified serum by an alloantibody present in normal sera. This is the basis for the Ham test. Therefore, the acronym HEMPAS (hereditary erythroiblastic multinuclearity with positive acidified serum) is commonly used as a synonym for CDA II. This differentiates it from paroxysmal nocturnal hemoglobinuria (PNH) where the test is positive with autologous sera. 14 SDS-PAGE of erythrocyte membrane proteins reveals a narrow and faster-migrating band 3 (anion exchange protein). A Western blot of membrane proteins shows the presence of reticulum-endothelial proteins (calreticulin, glucose-regulated protein 78, protein disulphide isomerase). 12 In CDA III, the clinical presentation is similar to CDA I and CDA II, but the anemia is never severe and the patients are not transfusion-dependent. Splenomegaly is only seen in some cases. The most striking abnormality in bone marrow smears is the presence of giant multinucleated erythroblasts containing up to 10 nuclei. 15 Additional features include abnormalities of retina with angioid streaks, macular degeneration, and a very high incidence of lymphomas and monoclonal gammopathies with or without myeloma. 16 Congenital dyserythropoietic anemia variants include a group of patients where the overall features cannot be attributed 5. The exact prevalence and frequency of this disease remains unknown due to the heterogeneity of clinical manifestations. According to available estimations, the incidence of CDA I and CDA II does not exceed 1 out of 100,000 births/year, while CDA III is extremely rare. Cases have been reported from Germany, France, Italy, Turkey, Spain, Switzerland, the Czech Republic, Romania, India, and Israeli Bedouins. 18 Etiology is unknown for all forms of CDAs. Transmission is recessive for CDA I and CDA II but dominant for CDA III. The gene for CDA I (CDAN1) is localized to chromosome 15q15.1 q15.3, and it codes for codanin 1, which may have a role in maintaining the integrity of the red cell nuclear membrane. Genes for CDA II (CDAN2) and CDA III (CDAN3) are localized to chromosomes 15q21 q25 and 20q11.2, respectively. 19,20 Anemia in CDA results from the combination of ineffective erythropoiesis and peripheral hemolysis. Red cell glycosylation defects are responsible for both of these mechanisms. 21,22 In CDA I, mutations of the codanin 1 gene result in the arrest of DNA synthesis in the S-phase. This cell-cycle abnormality probably induces multinuclearity and maturation discrepancies between the nucleus and cytoplasm, which, in turn, could cause enhanced intramedullary death and morphological abnormalities. The absence of codanin may be lethal, whereas 35% is compatible with life but is associated with severe clinical manifestations. 22 In CDA II, a narrow band 3 on SDS-PAGE indicates the insufficient incorporation of lactosaminoglycans in the cell membrane due to abnormal processing of N-glycans as a result of reduced activity of N- acetylglucosamyltransferase, alpha-mannosidase II, and membrane-bound galactosyltransferase. Lack of this carbohydrate moiety may increase hydrophobicity and induce clustering of membrane glycoproteins, which interferes with functions required for intracellular transport and compartmentalization, cell division, and differentiation Management of CDA is essentially symptomatic and includes hematological monitoring and surveillance of iron status. The decision to treat these cases depends on the age of presentation, severity of expression, type of CDA, and the presence of comorbid conditions. About 10% of patients require regular blood transfusion in infancy and childhood. 3 Approximately one third of patients with CDA I do not require blood transfusions after their first year of age but most develop iron overload even without regular transfusions. In transfusion-dependent cases, interferon-a remains the treatment of choice, since it is effective in improving chronic anemia and reducing ineffective erythropoiesis as well as normalization of progressively increasing serum ferritin. Thus, early diagnosis in the neonatal period is important. 6 In patients with CDA II, splenectomy is beneficial. It normalizes shortened red cell survival, produces a sustained increase in hemoglobin concentration, and decreases bilirubin levels, thus reducing transfusion requirements. However, it does not prevent further iron loading. 23,24 In the past, prednisolone, cobalamin, folate, and erythropoietin have been Downloaded 408 from LABMEDICINE j Volume Number 7 j July 2008 labmedicine.com

5 tried, but these do not have evidence of long-term efficacy. Allogeneic bone marrow transplantation was successful in some cases. 23,25 7. The major complications in CDA result from consequences of anemia or secondary hemosiderosis. 21 Ineffective erythropoiesis and increased peripheral red-cell destruction lead to iron overload, cholelithiasis, and splenomegaly. Secondary hemosiderosis may result in growth retardation, liver cirrhosis, heart failure, diabetes, hypothyroidism, or hypogonadism. 22 In order to prevent complications, confirmation of the diagnosis of CDA must be made in the first year of life. Adequate treatment with phlebotomies and/or iron chelating agents is helpful when the levels of ferritin approach 1,000 ng/ml. When a case of CDA is diagnosed, it is important to identify siblings who may also be affected. Counseling the parents, planning lifelong therapy, and followup are important parts of management. 26 In order to recognize diseasespecific complications and to avoid unnecessary diagnostic procedures, patients with CDA should be treated by hematologists, preferably in specialized centers. LM Keywords: congenital anemia, pediatric hematological diseases, dyserythropoiesis 18. Iolascon A, Servedio V, Carbone R, et al. Geographic distribution of CDA-II: Did a founder effect operate in Southern Italy? Haematologica. 2000;85: Tamary M, Shalmon L, Shaler H, et al. Localization of gene for congenital dyserythropoietic anemia to 15q Am J Hum Genetics. 1998;62: Gasparini P, Miraglia del Giudice E, Delaunay J, et al. Localization of the congenital dyserythropoietic anemia II locus to chromosome 20q11.2 by genome wide search. Am J Hum Genet. 1997;61: Queisser W, Spiertz E, Jost E, et al. Proliferation disturbances of erythroblasts in congenital dyserythropoietic anemia type I and II. Acta Haematol. 1971;45: Barosi G, Cazzola M, Stefanelli M, et al. Studies of ineffective erythropoiesis and peripheral haemolysis in congenital dyserythropoietic anaemia type II. Br J Haematol. 1979;43: Marchetti M, Quaglini S, Barosi G. Prophylactic splenectomy and cholecystectomy in mild hereditary spherocytosis: Analyzing the decision in different clinical scenarios. J Intern Med. 1998;244: Heimpel H, Eggl C, Griesshammer A, et al. Splenectomy in congenital dyserythropoietic anemia [abstract]. Egypt J Haematol. 2002;27: Iolascon A, Sabato V, De Mattia D, et al. Bone marrow transplantation in a case of severe, type II congenital dyserythropoietic anaemia (CDA II). Bone Marrow Transplant. 2001;27: Cazzola M, Barosi G, Bergamaschi G, et al. Iron loading in congenital dyserythropoietic anaemias and congenital sideroblastic anaemias. Br J Haematol. 1983;54: Heimpel H, Wendt F. Congenital dyserythropoietic anemia with karyorrhexis and multinuclearity of erythroblasts. Helv Med Acta. 1968;34: Iolascon A, Delaunay J, Wickramasinghe SN, et al. Natural history of congenital dyserythropoietic anemia type II. Blood. 2001;98: Iolascon A, De Mattia D, Perrotta S, et al. Genetic heterogeneity of congenital dyserythropoietic anemia type II [letter]. Blood. 1998;92: Heimpel H, Wendt F. Congenital dyserythropoietic anemia with karyorrhexis and multinuclearity of erythroblasts. Helv Med Acta. 1968;34: Boogaerts MA, Verwilghen RL. Variants of congenital dyserythropoietic anemia: An update. Haematologica (Budapest). 1982;15: Shalev H, Kapleushnik Y, Haeskelzon L, et al. Clinical and laboratory manifestations of congenital dyserythropoietic anemia type I in young adults. Eur J Haematol. 2002;68: Parez N, Dommergues M, Zupan V. Severe congenital dyserythropoietic anemia type I: Prenatal management, transfusion support and alpha-interferon therapy. Br J Hematol. 2000;110: Kato K, Sugitani M, Kawataki M. Congenital dyserythropoietic anemia type I with fetal onset of severe anemia. J Pediatr Hematol Oncol. 2001;23: Heimpel H. Congenital dyserythropoietic anemias: Epidemiology, clinical significance, and progress in understanding their pathogenesis. Ann Hematol. 2004;83: Gasser C. Congenital-dyserythropoietic-malformation syndrome (abstract). 6th meeting of the International Society of Hematology, Athens Heimpel H, Schwarz K, Ebnother M, et al. Congenital dyserythropoietic anemia type I (CDA I): Molecular genetics, clinical appearance and prognosis based on long term observation. Blood. 2006;107: Scartezzini P, Forni GL, Boldi M, et al. Decreased glycosylation of band 3 and band 4.5 glycoproteins of erythrocyte membrane in CDA. Br J Haemat. 1982;51: Wickramasinghe SN. Congenital dyserythropoietic anaemias: Clinical features, haematological morphology and new biochemical data. Blood Rev. 1998;12: Crookston JH, Crookston MC, Rosse WF. Red-cell abnormalities in HEMPAS (hereditary erythroblastic multinuclearity with a positive acidifiedserum test). Br Haematol. 1972;23: Wickramasinghe SN, Walilin A, Anstee D, et al. Observations on two members of Swedish family with congenital dyserythropoietic anemia III. Euro J Haemat. 1993;50: Delaunay J, Iolascon A. The congenital dyserythropoietic anaemias. Baillieres Best Pract Res Clin Haematol. 1999;12: Benjamin JT, Rosse WF, Dalldorf FG, et al. Congenital dyserythropoetic anemia: Type IV. J Pediatr. 1975;87: Downloaded labmedicine.com from July 2008 j Volume 39 Number 7 j LABMEDICINE 409