| Case Presentation | No. 18-2018 | A 10 month old girl with fever and cough

Mohammad Mizanur Rahman, Sifat-E-Moin and Debashish Saha

Article Info Presentation of Case Feeding history is also good and taking all types of solid and liquid foods. The parents are Department of Pathology, Combined Dr. Sifat-E-Moyen ( resident): A 10 also apparently healthy, the father is working in Military Hospital and Army Medical College Chattogram, Chattogram Can- month old baby girl, hailing from Cumilla (a an industry and mother is a housewife. Her tonment, Chattogram, Bangladesh city in the Chattogram Division), Bangladesh maternal and paternal uncles and aunts are also (MMR); Department of Pathology, was brought to the Department of Hematology, apparently healthy. With such findings, I made Armed Forces Medical Institute, Dhaka Armed Forces Institute of Pathology, Dhaka a provisional diagnosis and for proper Cantonment, Dhaka, Bangladesh (SEM); Department of Chemical Pathology, Cantonment, Dhaka, Bangladesh with the diagnosis, I advised to repeat complete blood Armed Forces Institute of Pathology, complaints of fever and cough for the last seven count, peripheral blood film examination, Dhaka Cantonment, Dhaka, Bangladesh days. With these complaints, the parents hemoglobin electrophoresis and reticulocyte (DS) initially took the child to a local child specialist. count. The child specialist gave initial supportive For Correspondence: Mohammad Mizanur Rahman medications and advised to investigate with the [email protected] , urine routine and

microscopic examination and Widal test. Provisional Diagnosis Received: 15 August 2018 Accepted: 28 August 2018 After 2 days, the parents again took the baby to under evaluation Available Online: 2 September 2018 the child specialist for reviewing the child and to show the investigations result. The child and ISSN: 2224-7750 (Online) all investigation’s results were reviewed by the Differential Diagnosis 2074-2908 (Print) child specialist and again advised to do Dr. Mohammad Mizanur Rahman: While cons- DOI: 10.3329/bsmmuj.v11i3.37995 hemoglobin electrophoresis. The parents did the hemoglobin electrophoresis and showed it tructing the differential diagnosis, I considered to the child specialist. After reviewing the the age of the child, recurrent mild illness Keywords: Anemia; Cough; Fever; Iron hemoglobin electrophoresis, the child specialist during the neonatal period and childhood and deficiency referred the case to a pediatric hematologist for socio-economic status. As there was no abnor- Cite this article: further management. mality other than the mild anemia, the follow- Rahman MM, Moin SE, Saha D. A 10 ing differential diagnoses were considered. month old girl with fever and cough. With the medications prescribed by the child Bangabandhu Sheikh Mujib Med Univ J. specialist of Cumilla, the child got cured but as Iron deficiency anemia 2018; 11: 246-255. the child repeatedly got sick after neonatal Among the global health problems, iron period, so the parents with anxiety and tension deficiency and iron deficiency anemia are the took the child to a pediatric hematologist in Copyright: leading issues encountered in everyday clinical The copyright of this article is retained Dhaka for better management and treatment. practice. The prevalence of iron deficiency by the author(s) [Atribution CC-By 4.0] Pediatric hematologist requested the parents to anemia is decreasing gradually but still, it is the repeat complete blood count and hemoglobin top-ranking cause of anemia worldwide and Available at: electrophoresis. Therefore, the parents did these has the significant sequel on the lives of young www.banglajol.info tests in the Armed Forces Institute of Pathology children and post-menopausal women in both 1 A Journal of Bangabandhu Sheikh Mujib where the concerned hematologist interviewed low-income and developed countries. Iron Medical University, Dhaka, Bangladesh the parents and examine the baby. On general deficiency anemia may occur at any age but in examination, mild anemia was found, her mile- infancy and childhood, it is one of the most stones of development were age proportional, common forms of anemia. The greatest preva- , clubbing, koilonychia, angular lence is in between the ages of 6 and 24 months stomatitis were absent and there were no but may occur up to the age of 5 years. Iron bleeding manifestations. All other parameters deficiency anemia is rather infrequent in school were within normal limit. Systemic -going children.2 Iron is an essential and pivotal examinations revealed no abnormality as well element that is required in several biologic as and hepatosplenomegaly functions such as respiration, energy produc- were absent. Vaccination was done as per the tion, DNA synthesis and cell proliferation.3 national immunization schedule. The baby was Human body has developed to preserve the delivered per vaginally normally and she is the iron in a number of ways such as the recycling only child of the non-consanguineous parents. of iron after the breakdown of red cells and BSMMU J 2018; 11: 246-255 247

part of the duodenum as well as pH and redox Table I potential of this part of the intestine favors iron absorption. For the absorption of non-heme and Hematological parameters of the patient and her parents heme iron, at least three pathways are known. Absorption of non-heme iron utilizes divalent metal Patient Father Mother transporter 1 (ferrous valence) and/or mobilferrin- Hemoglobin (g/dL) 8.5 11.4 12.3 integrin-paraferritin [ferric valence] and heme iron count (x1012/L) 4.0 5.3 4.1 is absorbed through the third pathway utilizing MCV (fL) 58.7 65.3 82.3 heme oxygenase. Iron absorption depends on the body’s need for iron. Active erythropoiesis and/or MCH (pg) 23.5 25.1 29.7 iron deficiency increases iron absorption but on the MCHC (g/dL) 21.1 24.8 31.5 other hand, iron overload down-regulates its Red cell distribution width–CV 14.8 15.2 14.9 absorption. Iron absorption involves two notable Reticulocyte (%) 1.3 1.5 1.9 steps: Mucosal uptake of iron and transfer of this iron from mucosal cell to the lamina propria where Hb A 75.6 75.5 97.3 it enters into the plasma. Specific iron carrier Hb A2 1.4 1.9 2.7 proteins are required in both steps.2 Excess free iron HbF - - - are toxic to the human body and participate in Fenton reaction to generate free radicals that cause Hb Bart’s 23.0 22.6 - oxidative cell damage, that’s why its absorption is retention of iron in the absence of an excretion limited to 1–2 mg daily. mechanism. The physiology of iron homeostasis is The daily iron requirement is about 25 mg and most now well-understood and involves a number of are provided through recycling by macrophages proteins including duodenal ferric reductase, that senescent erythrocytes.4, 5 Both iron divalent metal transporter 1, ferroportin, absorption and release from the body stores are hephaestin, transferrin receptor 1 and 2, tightly controlled by a hormone, hepcidin.3 Hepci- hemochromatosis factor and hemojuvelin. All such din is a hormone synthesized by the and plays proteins are expressed at highest levels in the first a vital role in iron homeostasis. It also acts as an acute phase protein that acclimatizes the variation A of plasma iron level. During iron metabolism, hepcidin binds to cell-surface ferroportein, causing its degradation in lysosomes. Therefore, hepcidin inhibits iron absorption, iron release from macro- phages and iron transport across the placenta.6 Raised plasma hepcidin level is found in hemo- chromatosis, patients with systemic inflammation or infections. Its level is reduced or undetectable in iron deficiency anemia, erythroid hyperplasia and tissue hypoxia. ELISA or mass-spectrometry-based techniques are employed to measure serum or urinary hepcidin.7 During the third trimester of pregnancy, 80% of the iron present in the newborn term infant is accumu- lated from the mother. Therefore, premature infants B skipped this accretion of iron and are lack of total body iron stores. Besides this, several maternal factors such as anemia, maternal hypertension with intrauterine growth retardation and gestational diabetes also contribute to low iron stores in both term and pre-term infants.8 The Institute of Medi- cine recommended the daily iron allowance for 7 to 12 months’ completed age is 11 mg/day. From birth to 6 months’ completed age, iron requirement is less in comparison to infants after 6 months’ age because of rapid growth and increased demand.9 Anemia is labeled for both male and female children aged 12 months through 35 months, when hemoglobin level is less than 11 g/dL as per the guidelines of US Figure 1: Hemoglobin patterns of the patient (A) and father (B) National Health and Nutrition Survey 1999–2002. 248 BSMMU J 2018; 11: 246-255

When the amount of iron in the body is not iron in breast milk is high in comparison to the sufficient to preserve normal physiologic function, growth of the child, it is inadequate. During the first then the state is defined as iron deficiency. Iron 6 months, infants use the iron from their iron stores deficiency may or may not be accompanied by iron and then additional iron is required from foods for deficiency anemia. Anemia associated with iron normal erythropoiesis in the subsequent period.15 deficiency is called iron deficiency anemia.10 At Solid foods rich in iron should be given after 6 present, there is no national statistics regarding the months to maintain normal erythropoiesis. Foods incidence of iron deficiency and iron deficiency such as meat, fish, eggs and green leafy vegetables anemia among infants before the age of 12 months. are rich in iron and such foods should be given to According to the Institute of Medicine report, the infants after the age of 6 months to meet the iron prevalence of iron deficiency at 6 months of age is requirement. Excessive consumption of cow’s milk 4% and increased to 12% at 12 months of age.11 by the infants may cause chronic blood loss due to Anemia is one of the most important pointers of heat-sensitive protein present in the cow’s milk and iron deficiency or iron deficiency anemia. Never- presence of calcium as well as casein phospho- theless, without anemia, the iron deficient state may peptides in cow’s milk impairs iron absorption develop and in such situation, tissues may suffer resulting in iron deficiency anemia.12 However, a from hypoxia. Iron deficiency anemia develops in commonly accepted generality is that dietary three stages: Pre-latent stage which is marked by deficiency alone is not sufficient to cause a clinically reduced iron stores due to less intake of iron. At this significant iron deficiency, therefore attempt should stage hemoglobin level may remain normal for a always be made to find out a source of blood loss as while indicating the presence of iron deficiency in a cause of iron deficiency after the age of 1 year.16 the absence of anemia. During this time, serum iron In general, anemia produces non-specific of is normal but only plasma ferritin level and plasma the mucous membrane. Specific to iron deficiency transferrin saturation are reduced. This is followed anemia are epithelial tissue abnormalities such as by a latent stage where iron stores are depleted but esophageal webbing, koilonychias, glossitis, angular still blood hemoglobin level is normal. Biochemical stomatitis and gastric atrophy. The relationship parameters such as plasma ferritin level and plasma between iron deficiency anemia and such epithelial transferrin saturation are reduced, total iron changes is not clear.17 History and physical binding capacity is increased. The next stage is the examination may elicit the etiology of iron development of iron deficiency anemia which is deficiency anemia but laboratory investigations marked by low hemoglobin level and negative iron such as complete blood count, peripheral blood film balance indicated by low plasma iron, plasma study, serum iron, total iron binding capacity and transferrin saturation and increased total iron serum ferritin are usually sufficient to establish an binding capacity. Iron deficiency anemia is the most uncomplicated iron deficiency anemia. Evaluation common nutritional deficiency anemia in infancy of hemoglobin electrophoresis, measurement of and in adolescents who have menstruation but hemoglobin A2, red cell distribution width (RDW) those having increased growth rate and cannot meet and hemoglobin content of reticulocyte are helpful their requirements are at increased risk of to differentiate from other .18 Age developing iron deficiency anemia.2, 12 Maternal of the patient and presence of anemia favors the iron crossing through the placenta is the sole source diagnosis of iron deficiency anemia in this child but of iron for the fetus in utero. Full-term fetus age proportional development, the absence of contains iron in about 75 mg/kg body weight in the features such as angular stomatitis, koilonychia and last trimester of pregnancy and such iron stores are history of taking iron-rich foods are the points adequate enough for erythropoiesis in the initial 6 against the diagnosis of iron deficiency anemia. months of life. In children, the etiologies of iron Acquired deficiency anemia are insufficient intake with rapid growth, low birth weight and gastrointestinal losses Increased red cell destruction may result from due to exorbitant consumption of cow’s milk. In either inherited or acquired disorders. Hemolytic low birth weight infants, the iron stores are anemia results when compensatory increased exhausted earlier as they are smaller. In such cases, erythropoiesis by the bone marrow is unable to the risk of iron deficiency anemia can be reduced by recompense increased red cell destruction. The delayed clumping of the umbilical cord after common causes of acquired hemolytic anemia from delivery.13 Iron content in breast milk is highest in infancy to toddlerhood are: -mediated the first month and then reduces gradually in the , hemolytic uremic syndrome, drug- ensuing periods and reaches to 0.3 mg/L in about induced hemolysis and disseminated intravascular fifth month.14 However, iron content in breast milk hemolysis.19 Antibody-mediated hemolysis is the cannot be increased by providing iron-rich foods to most important etiology of acquired hemolytic the mother but breast milk’s iron absorption is high anemia. Two types of such as auto- (50%) in comparison to food iron. Also, absorption antibodies and alloantibodies are usually respon- of iron in breast milk is reduced if given with other sible for the development of antibody-mediated foods to infants. Therefore, though absorption of hemolytic anemia. are produced by BSMMU J 2018; 11: 246-255 249 the patient’s own immune system and directed directed against the epitopes of Rh system and react against epitopes of the patient’s own red cell at 37°C, liable for the warm type of AIHA. antigens.20 Characteristic features of the auto- Occasionally IgA auto-antibodies are generated and antibody determine the type of AIHA. Ig class of usually associated with IgG, rarely operative as the the and thermal features i.e., the single etiology of AIHA.27 IgM autoantibodies are optimal temperature of the reaction at which also formed in this disease causing mainly intra- autoantibody reacts with autologous red cells vascular hemolysis as these antibodies are penta- divided AIHA into three types such as warm AIHA, meric, capable of fixing complement more efficient- cold disease (CHAD) and mixed ly but extravascular hemolysis mainly in the liver forms.21 AIHA may also be classified as primary occurs when it is C3d mediated. Autoantibodies of (idiopathic), in which red cell destruction occurs in these type are directed against I/i blood group the absence of any other coexisting disorder, or as a system and react optimally at 4°C, categorizing it as secondary form in which hemolysis occurs secon- a cold form of AIHA. Nonetheless, the range of the dary to an underlying disease. Warm AIHA is thermal amplitude of IgM autoantibodies is 0–34°C. usually idiopathic or secondary to other autoimmu- If the thermal amplitude of IgM autoantibodies is ne disorders. Acute CHAD are mainly secondary to close to physiological temperature (Warm IgM), infections while chronic CHAD is predominantly then the severe form of AIHA ensures in which associated with lymphoproliferative or neoplastic cases the mortality rate may be 18–25%.25 Intra- diseases.22 Genetic predisposition and environmen- vascular hemolysis in AIHA is more detrimental tal factors are important in the pathogenesis of and severe than extravascular hemolysis as the rate . Several autoimmune diseases have of RBC destruction is 200 mL per hour in case of HLA association but still no HLA linkage has been intravascular hemolysis which is 10 times more identified in AIHA. Mechanisms involving the than extravascular hemolysis.22 breakdown of central and peripheral tolerance in AIHA may occur after allogeneic hemopoietic stem the pathogenesis of AIHA are substantially cell transplantation. The 3 year cumulative inci- unknown.23 Another mechanism of autoimmunity dence in such a population is about 4.4%.28, 29 AIHA is molecular mimicry involving self-antigens and is rare with an estimated incidence of 0.2 per million foreign pathogens. Most probably this mechanism individuals below 20 years of age.30 Peak incidence is mainly responsible in the pathogenesis of AIHA is found in less than four years of age.31 The secondary to various bacterial, or virus mortality rate is approximately 11%.32 Most patients infections likely due to the presence of common with AIHA presents their clinical manifestation antigenic epitopes on red cell proteins or insidiously but some patients have severe acute carbohydrates. Polyclonal lymphocyte activation symptoms. Symptoms may range from caused by several viral infections and the asymptomatic compensated reticulocytosis with emergence of “forbidden clones” in lymphoproli- mild hyperbilirubinemia to acute fulminant ferative disorders are more other mechanisms of hemolysis leading to jaundice, hepatosplenomegaly, development of AIHA.24 Another form of AIHA is tachycardia and angina. They are also determined drug-induced AIHA where environmental agents by the causative factor responsible for AIHA and or drugs take part in the development of neo- degree of hemolysis which again depends on the antigens which stimulate antibody production type of autoantibody. IgM warm reactive AIHA against red cells culminating the destruction of presents with life-threatening acute intravascular RBCs until the autoimmune response is destroyed hemolysis with high mortality rate compared to and removed from the body.25 Autoantibodies patients with cold type AIHA where the symptoms against red cells have also been detected in about are usually mild.33 Bone marrow compensatory 30% of individuals who previously received red cell ability also determine the degree and severity of transfusion for any reason where transfused red anemia. Those patients with AIHA who respond cells elicit the generation of autoantibodies.26 Many their anemia by reticulocytosis usually presents other hypotheses about autoimmune/inflammatory with less severe clinical symptoms than patients diseases have come to the attention in the recent who do not respond by reticulocytosis rather years but the most agreed mechanism is the loss of develop . Such reticulocytopenia supperssor mechanisms and breakdown or are observed in 20% of adult and 38% of children alteration of self-tolerance.23 with AIHA.34 Laboratory diagnosis depends on the IgG is the most common antibody formed against detection of autoantibody either by direct antiglo- red cells in AIHA and causes extravascular hemo- bulin test (DAT) or column or flow lysis through antibody-dependent cell mediated cytometry. Positive DAT is found with anti-IgG cytotoxicity (ADCC) in the reticuloendothelial sys- (usually in warm AIHA) and/or anti-C3d (usually tem especially in the . Red cell breakdown is in cold AIHA) antisera. About 10% of all cases of influenced by the subclass of IgG; IgG1 together AIHA due to anti – IgA, low-affinity IgG or RBC- with IgG3 shortens the half-life more effectively bound IgG below the detection threshold of the test than IgG2 and IgG4. These autoantibodies are may yield false negative DAT. Therefore, not all 250 BSMMU J 2018; 11: 246-255

AIHA cases show positive DAT results. To diagnose ficant role. The anemia in chronic inflammation is the AIHA with false negative DAT, more sensitive usually normocytic normochromic and refractory to reagents such as mono-specific anti-IgA antisera/ therapy with hematinics.37 The dominant under- low ionic strength solutions (LISS) and techniques lying mechanism is impaired response of the bone such as column agglutination test or flow cytometry marrow to erythropoietin (EPO) due to disturbance can be considered. Supportive laboratory investiga- of iron metabolism which is mediated by a recently tions for AIHA may include estimation of serum discovered peptide hormone called hepcidin.38-40 lactate dehydrogenase (LDH), reticulocyte count Hepcidin is synthesized in the liver, present in and examination of blood film which shows a plasma and excreted through urine and functions as significant number of spherocytes.35 In addition to a major regulatory hormone for iron transport.41 traditional treatment by and immu- Hepcidin controls the activity of ferroportin 1, a nosuppressive drugs as first-line therapy or sple- transmembrane domain protein present on the nectomy as second-line therapy, recently few emer- surface of enterocytes, hepatocytes and macr- ging treatment modalities are considered which phages. The movement of iron through these cells’ include administration of (anti-CD20 wall is regulated by ferroportein 1. By binding with monoclonal antibody), erythropoiesis-stimulating ferroportein 1, hepcidin inhibits the function of agents, other immunosuppressive agents such as ferroportein 1 and thereby prevents the transpor- cyclosporine A (CsA), danazol (synthetic anabolic tation and exit of iron through the cell membrane. steroid) and showed meaningful success. These As a consequence, mobilization from hepatocytes therapies especially rituximab can be applied in and release of sequestered iron from macrophages patients who are refractory to /sple- is impaired and result in a decrease in the nectomy or relapse after splenectomy. Cortico- availability of iron for erythropoiesis. In inflamma- steroid as the first-line treatment for patients with tory states, hepcidin synthesis is increased mainly warm AIHA provides a response rate of 70–85%. by the cytokine IL- 6 and thus preventing the body Rest of the patients still needs second-line therapy. from more iron absorption and inhibiting iron On the other hand, due to the presence of co- transportation. Hence, the response of anemia of inflammation to EPO and EPO stimulating agents is existence morbid conditions such as diabetes, suboptimal.42 Anemia of inflammation is an anemia uncontrolled hypertension, obesity, osteoporosis, that develops within 1–2 months of the onset of peptic ulcer or children where treatment with illness. Anemia persists without increasing in seve- corticosteroid is relatively contraindicated and in rity. Chronic pulmonary infections such as pulmo- such cases second-line therapy i.e., splenectomy is nary tuberculosis, pulmonary abscess, subacute thought to be the most effective and produce high bacterial endocarditis, pelvic and urologic infec- response rates up to 82%. Rituximab (so-called tions, osteomyelitis, chronic fungal disease, menin- "medical splenectomy") is another effective second- gitis and human immunodeficiency virus infections line therapy, providing high initial response rates are the predominant clinical conditions which up to 85% and prolonged disease-free survivals of predispose to the development of anemia of infla- 55% up to 2 years with reduced adverse reactions.36 mmation.43-45 The anemia is usually mild and In this patient, age, presence of anemia and possible morphologically either normocytic normochromic viral respiratory tract infections favors the diagnosis or microcytic . The clinical of AIHA but chronic as well as less severe anemia features are those of the underlying disorders.46 for a longer duration are the points against the Anemia of inflammation is diagnosed by the pre- diagnosis of AIHA. sence of anemia in association with inflammatory or Concurrent infections infectious disease. Laboratory investigations reveal- ed low serum iron, decreased soluble transferrin Anemia is one of the most noticeable features of receptors and reduced transferrin saturation with infection. The severity of anemia in infection ranges iron. Reticuloendothelial iron stores are increased from a mild, asymptomatic form to an acute life- depicted by Perl stain of bone marrow sample and threatening complication of infection. Anemia of increased serum ferritin.47 Anemia of inflammation chronic disease is defined as the anemia seen in is usually treated by alleviating the underlying patients with chronic infections. In addition to disorders. Iron therapy is unsatisfactory unless infections, anemia is also seen in other chronic there is a concomitant iron deficiency. Administra- diseases including and chronic inflammatory tion of EPO may provide mild or suboptimal disorders such as rheumatoid arthritis, inflamma- response.37 tory bowel disease and renal failure. This anemia is referred to as anemia of inflammation. In acute This girl has been suffering from upper respiratory inflammation, anemia is directly related to bone tract infection and fever which favor the diagnosis marrow suppression and acute hemolysis but in of anemia of inflammation but presence of weak- chronic inflammation, several factors are involved ness from toddlerhood; less playful and persistent in the pathogenesis of anemia. Inflammatory cyto- mild anemia are the points against the diagnosis of kines, hepcidin and bacterial toxins play a signi- anemia of inflammation. BSMMU J 2018; 11: 246-255 251

Congenital hemolytic anemia Dr. Moin: Keeping in mind the diagnosis of congenital hemolytic anemia, especially Premature destruction of red cells is called hemo- and in this girl, I advised the lysis. Mild hemolysis is not manifested as anemia parents to do some laboratory investigation of the but if the erythrocyte loss is more than the whole family members (patient, his father and compensatory activity of the bone marrow then mother) such as complete blood count, blood film only anemia develops which may be of varying examination, reticulocyte count and capillary hemo- severity.48 There are a wide range of causes, both globin electrophoresis. The results of all investiga- congenital and acquired, responsible for hemo- tions are shown in Table I. Capillary hemoglobin lysis.49, 50 Red cell membrane, enzyme and electrophoresis showed a most remarkable finding hemoglobin defects are the main etiologies of and revealed 23.0 and 22.6% Hb Bart’s in zone 12 of congenital hemolytic anemia. In congenital hemo- the electrophoretic graph of the patient and father lytic anemia the genes that master the production of respectively which is shown in Figure 1. After red cells are defective. On the basis of the affected getting such finding I advised to do HbH inclusion defective genes, different types of congenital test to differentiate from HbH. But the test turned hemolytic anemia develop. In each type, abnormal out as negative in both the cases which almost erythrocytes are produced in the body and destroy- confirms that it is Hb Bart’s not HbH. ed prematurely.51 Hemolytic anemia in children should be suspected in any child with anemia, jaundice and . In severe cases, skeletal deformities especially of the skull and facial bones Discussion due to erythroid hyperplasia and due Dr. Rahman: Recent introduction of capillary to high cardiac out may be demonstrated by hemoglobin electrophoresis in the diagnosis of radiographs. A few patients may also develop pig- congenital hemolytic anemia facilitates the precise ment gallstones after the first decade of life. detection of different types of abnormal hemoglobin Children with mild hemolytic anemia may have than by gel hemoglobin electrophoresis used in the nearly normal hemoglobin levels if reticulocyte recent past. By exploiting this capillary hemoglobin production increases sufficiently for the increased electrophoresis method, it enables us to detect Hb rate of RBC destruction. However, concurrent infec- Bart’s in zone 12 of electrophoretic graph in these tious illness may aggravate the anemia during two patients which is absolutely distinct from which mild hemolytic cases can be identified. hemoglobin H which appears in zone 15 of the Hemoglobin level falls during infectious illness due graph. Hb Bart’s is detected in a small amount of 1– to oxidative stress and depression of bone marrow 2% in newborn infants with alpha thalassemia trait activity.52 Examination of blood film is the mainstay and 5–6% in alpha thalassemia carrier.55 in the evaluation of hemolytic anemia and in some cases, the morphology of red cells may point to the Genetically the different types of alpha thalassemia diagnosis of a typical congenital hemolytic anemia. occur on the basis of the number of alpha However, it is also important to keep in mind that thalassemia gene(s) a person is missing. Missing similar morpho-logical features may be found in one alpha thalassemia gene (αα/α -) indicates silent acquired hemolytic anemia such as spherocytes alpha thalassemia carriers or two alpha thalassemia which are found not only in hereditary sphero- genes (α-/α– or αα/- -) indicates alpha thalassaemia cytosis but also in immune hemolytic , ABO trait. Individuals with either silent alpha thala- incompatibility in neonates and other forms of ssemia carrier or alpha thalassemia trait are asymp- oxidant damage to the red cell membrane.53 After a tomatic with normal laboratory features. On the critical evaluation of blood film, further investiga- other hand, the number of dysfunctional genes is tions such as reticulocyte count, hemoglobin elec- reflected by the percentage of Hb Bart’s seen on the trophoresis, estimation of HbA2, G6PD estimation, newborn screen by capillary hemoglobin electro- osmotic fragility test, hemoglobin insolubility test, phoresis. Presence of less than 25% Hb Bart’s sickling test and Coomb’s test may be advised to indicates loss of one (silent carrier) or two (alpha confirm the diagnosis.54 thalassaemia trait) genes. Genetically deletion or dysfunction of three of the four alpha globin genes Presence of respiratory tract infections, fever and manifests as HbH disease and deletion or dysfunc- absence of splenomegaly disfavor the diagnosis of tion of all of the four alpha globin genes leads to Hb congenital hemolytic anemia but the presence of Bart’s hydrops foetalis where the percentage of Hb from toddlerhood, less playful, persistent Bart’s is 80% or more and the affected infants are mild anemia after recovery from respiratory tract either born dead or die within a few hours of infection and age of the patient favor the diagnosis birth.55 of congenital hemolytic anemia. Alpha thalassemia is caused by mutations or deletions affecting either one or more α–globin Dr. Moin’s Diagnosis genes, leading to decreased or absent α–globin chain production from the affected gene(s).56 Congenital hemolytic anemia Insignificant hematologic findings are usually the 252 BSMMU J 2018; 11: 246-255

result of deletion or inactivation of only one α– thalassemia carrier or alpha thalassemia trait. globin gene. When two α–globin genes deleted or Dr. Imana: Did you exclude iron deficiency anemia inactivated, either both on the same chromosome 16 in this case as low HbA2 indicate the possibility of (in cis) or on one each of the two chromosomes 16 iron deficiency anemia? (in trans), the affected person is well but has borderline anemia, as well as microcytic hypochro- Dr. Monwar Tark: In this patient blood film showed mic red blood cells.57 no specific features such as elongated and pencil- shaped cells which are characteristic of iron Deletions removing one α–globin gene are results of deficiency anemia. Moreover, serum iron profile misalignment crossovers during meiosis. This single was done and it was within the reference limit. α–globin gene deletion is known as silent alpha thalassemia carrier. In silent alpha thalassemia, the Dr. Durdana Mahin: What is the prognosis of this remaining normal three α–globin genes can patient and how will follow up this case? compensate nearly completely and only a low level of Hb Bart’s is detected. These individuals are likely Dr. Rahman: The patient will remain asymptomatic to be clinically and hematologically normal.56 Silent unless affected by any conditions which suppress alpha thalassemia carrier is very common and the bone marrow. She should be followed-up by found in 30% of African Americans and up to 60 to doing hemoglobin level monthly and serum ferritin 80% of people living in parts of Saudi Arabia, India, six monthly. Thailand and Papua New Guinea.58 Dr. Arif Ahmed Khan: How will you confirm the Alpha thalassemia trait typically results from the diagnosis and have you done it? dysfunction of two α–globin genes, either due to Dr. Moin: By doing alpha thalassemia mutation gene deletions or a specific change in α–globin gene study, we can confirm the diagnosis. However, that produces elongated alpha globin. It produces family screening and pedigree study (Figure 2) moderate level of Hb Bart’s. These conditions are showed the presence of Hb Bart’s in her father and usually benign although mild to moderate these findings almost confirm the diagnosis of microcytic anemia is common. The patient in the either alpha thalassemia carrier or trait. I have not present case presentation and his father had also done the molecular diagnosis as it is not available in moderate level of Hb Bart’s (23.0 and 22.6%) and our country at present. mild to moderate anemia (8.5 and 11.4 g/dL) with microcytic hypochromic red cells. Therefore, the Dr. Elias: By quantifying Hb Bart’s by capillary amount of Hb Bart’s in the peripheral blood may be hemoglobin electrophoresis, how can we diagnose used to predict the genotype of α–thalassemia.59 different types of alpha thalassemia? Dr. Lutfunnahar Khan: What is the cut off value of Dr. Rahman: Presence of Hb Bart’s less than 25% Hb Bart’s for diagnosing alpha thalassemia trait or indicates the presumptive diagnosis of either alpha carrier? thalassemia carrier or alpha thalassemia trait. Hb Bart’s ≥ 25% indicates the diagnosis of HbH disease Dr. Moin: Two types of information regarding the in neonates.58 In Hb Bart’s hydrops fetalis, its quantity of Hb Bart’s are available. However, if the amount is approximately 80–85% which is Hb Bart’s level is less than 25%, the condition is incompatible with life.55 Here, I like to mention that likely to be either alpha thalassemia carrier or alpha there is no conclusive data regarding the amount of thalassemia trait. Here I want to mention that in Hb Bart’s when it is in between 25–80%, then what most published literates such cut off value of Hb should be nature of the condition. Therefore we Bart’s is given for neonates but here we found such recommend studying and analyzing alpha amount of Hb Bart’s both in the adult patient and thalassemia gene in more cases to resolve the his 10-month old daughter. queries when the amount of Hb Bart’s is in between 25 to 80% as no conclusive data is available in the Dr. Shourov: Here the HbA2 level is low. What are the conditions where HbA2 level is low? Unkonwn (not alive) Dr. Monirul Islam: Low HbA2 level is found mainly in iron deficiency anemia and alpha thalassemia. Normal male Delta and hereditary persistence of fetal hemoglobin are other conditions which may Affected male cause low HbA2. Normal female

Dr. Zannat: Did low HbA2 level in this case support Affected female the diagnosis?

Dr. Shahidul Islam: In this case low HbA2 level along Index case with the presence of moderate amount of Hb Bart’s strongly suggest the presence of either alpha Figure 2: Pedigree chart of the patient BSMMU J 2018; 11: 246-255 253 literature as well as published recognized books children and women between 1988-1994 and 1999- about the presence of Hb Bart’s in the mentioned 2002. Am J Clin Nutr. 2008; 88: 1611-17. range. 11. Dallman PR. Iron deficiency anemia: A synthesis of Dr. Debashish Saha: What type of advice should be current scientific knowledge and U.S. given to the parents? recommendations for prevention and treatment. In: Iron deficiency anemia: Recommended guidelines Dr. Rahman: Genetic counseling is most important for prevention, detection and management among advice in such inherited disorders as such U.S. children and women of childbearing age. Earl counseling reduces the reproductive risk of the R, Woteki CE (eds). Washington, National family. Academies Press, 1993, pp 41-97. 12. Ozdemir N. Iron deficiency anemia from diagnosis to treatment. Turk Pediatri Ars. 2015; 50: 11-19. Final Diagnosis 13. van Rheenen P. Less iron deficiency anaemia after Hemoglobin Bart’s (silent alpha thalassemia delayed cord-clamping. Paediatr Int Child Health. carrier/alpha thalassemia trait) 2013; 33: 57-58. 14. Siimes MA, Vuouri E, Kuitunen P. Breast milk iron: A declining concentration during the course of References lactation. Acta Paediatr Scand. 1979; 68: 29-31. 15. Celada A, Busset R, Gutierrez J. No correlation 1. McLean E, Cogswell M, Egli I, Wojdyla D, de between iron concentration in breast milk and Benoist B. Worldwide prevalence of anaemia, maternal iron stores. Helv Paediatr Acta. 1982; 37: WHO Vitamin and Mineral Nutrition Information 11-16. System, 1993-2005. Public Health Nutr. 2009; 12: 444-54. 16. Hempel EV, Bollard ER. The evidence-based evaluation of iron deficiency anemia. Med Clin 2. Saxena R, Pati HR, Mahanpatra M. de Gruchy’s North Am. 2016; 100: 1065-75. Clinical hematology in Medical Practice. In: Saxena R, Pati HR, Mahanpatra M, editors. 6th ed. New 17. Mateos GME, Cruz BJ, Lopez LE, Valdes SMD, Delhi. Wiley India Pvt. Ltd., 2013, p 33-57. Nogales EA. Review of hematology and biochemistry parameters to identify iron deficiency. 3. Hentze MW, Muckenthaler MU, Galy B, An Pediatr (Barc). 2009; 71: 95-102. Camaschella C. Two to tango: Regulation of mammalian iron metabolism. Cell 2010; 142: 24-38. 18. Goddard AF, James MW, McIntyre AS, Scott BB. Guidelines for the management of iron deficiency 4. Camaschella C. Iron-deficiency anemia. N Engl J anemia. Gut 2011; 60: 1309-16. Med. 2015; 372: 1832-43. 19. Oski FA, Brugnara C, Nathan DG. A diagnostic 5. Kumar V, Abbas AK, Aster JC. Robbins and Cotran approach to the anemic patient. In: Nathan and Pathologic Basis of Disease. In: Kumar V, Abbas Oski's Hematology of infancy and childhood. 6th AK, Aster JC (eds). 9th ed. Philadelphia, Elsevier ed. Philadelphia, Saunders, 2003, pp 409-18. Saunders, 2015, pp 47-48. 20. Gehrs BC, Friedberg RC. Autoimmune hemolytic 6. Camaschella C, Hoffbrand AV, Hershko C. Iron anemia. Am J Hematol. 2002; 69: 258-71. metabolism, iron deficiency and disorders of hem synthesis. In: Hoffbrand AV, Higgs DR, Metha AB, 21. Petz LD, Garratty G. Immune hemolytic anemias, editors. 7th ed. New Delhi, India. Wiley Blackwell, 2nd ed. Philadelphia, Churchill Livingstone, 2004. 2016, p 26-52. 22. Berentsen S. How I manage cold agglutinin disease. 7. Camaschella C. Iron and hepcidin: A story of Br J Hematol. 2011; 153: 309-17. recycling and balance. Hematol Am Soc Hematol Educ Program. 2013; 2013: 1-8. 23. Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology, Philadelphia, Saunders, 8. Rao R, Georgieff MK. Micro minerals. In: Nutrition 2012. of the preterm infant: Scientific basis and practical guidelines. Tsang RC, Uauy R, Koletzko R, Zlotikin 24. McQueen F. A B-cell explanation for autoimmune SH (eds). Cincinnati, Digital Educational disease: The forbidden clone returns. Postgrad Med Publishing Inc, 2005, pp 277-310. J. 2012; 88: 226-33. 9. Institute of Medicine. Dietary reference intakes for 25. Garratty G, Arndt PA. Drugs that have been shown vitamin A, arsenic, boron, chromium, copper, to cause drug-induced immune hemolytic anemia iodine, iron, manganese, molybdenum, nickel, or positive direct antiglobulin tests: Some silicon, vanadium, and zinc. Washington, National interesting findings since 2007. Immunohematology Academies Press, 2003. 2014; 30: 66-79. 10. Cusick SE, Mei Z, Freedman DS. Unexplained 26. Ahrens N, Pruss A, Kähne A, Kiesewetter H, decline in the prevalence of anemia among US Salama A. Coexistence of autoantibodies and 254 BSMMU J 2018; 11: 246-255

alloantibodies to red blood cells due to blood liver. J Biol Chem. 2001; 276: 7806-10. transfusion. Transfusion 2007; 47: 813-16. 42. Safran M, Kaelin WG. HIF hydroxylation and the 27. Sokol RJ, Hewitt S, Stamps BK. "Autoimmune mammalian oxygen-sensing pathway. J Clin Invest. haemolysis: An 18-year study of 865 cases referred 2003; 111: 779-83. to a regional transfusion centre”. Br Med J. 1981; 282: 2023-27. 43. Cash JM, Sears DA. The anemia of chronic disease: Spectrum of associated diseases in a series of 28. Mayer B, Yürek S, Kiesewetter H, Salama A. Mixed- unselected hospitalized patients. Am J Med. 1989; type autoimmune hemolytic anemia: Differential 87: 638-44. diagnosis and a critical review of reported cases. Transfusion 2008; 48: 2229-34. 44. Cartwright GE. The anemia of chronic disorders. Semin Hematol. 1966; 3: 351-75. 29. Sanz J, Arriaga F, Montesinos P, Ortí G, Lorenzo I, Cantero S, et al. Autoimmune hemolytic anemia 45. Cartwright GE, Lee GR. The anemia of chronic following allogeneic hematopoietic stem cell disorders. Br J Hematol. 1971; 21: 147-52. transplantation in adult patients. Bone Marrow 46. Chernow B, Wallner SF. Is the anemia of chronic Transplant. 2007; 39: 555-61. disorders normocytic-normochromic? Military 30. Singh A, Mandal A, Patel A, Mishra S. Med. 1978; 143: 345-46. Autoimmune Haemolytic Anaemia-A Spectrum of 47. Lee MH, Means R. Extremely elevated serum Presentation in Children. J Clin Diagn Res. 2017; 11: ferritin levels in a university hospital: Associated SR01-02. diseases and clinical significance. Am J Med. 1995; 31. Vaglio S, Arista MC, Perrone MP, Tomei G, Testi 98: 566-71. AM, Coluzzi S, et al. Autoimmune hemolytic 48. Gallagher PG. The red blood cell membrane and its anemia in childhood: Serologic features in 100 disorders: , elliptocytosis, cases. Transfusion 2007; 47: 50-54. and related diseases. In: Williams Hematology. 32. Zanella A, Barcellini W. Treatment of autoimmune Kaushansky K, Lichtman MA, Beutler E, Kipps TJ, hemolytic anemias. Haematologica 2014; 99: 1547- Seligsohn U, Prchal JT (eds). 8th ed. New York, 54. McGraw Hill, 2010, pp 617-46. 33. Bass GF, Tuscano ET, Tuscano JM. Diagnosis and 49. Gallagher PG. Red cell membrane disorders. In: classification of autoimmune hemolytic anemia. Hoffman R, Benz EJ, Silberstein LE, Heslop H, Autoimmun Rev. 2014; 13: 560-64. Weitz J, Anastasi J, eds. Hematology: Basic Principles and Practice. 6th ed. New York, NY: Churchill 34. Aladjidi N, Leverger G, Leblanc T, et al. New Livingstone, 2013; 592-613. insights into childhood autoimmune hemolytic anemia: A French national observational study of 50. Shah A. Acquired hemolytic anemia. Indian J Med 265 children. Hematologica 2011; 96: 655-63. Sci. 2004; 58: 533-36. 35. Thedsawad A, Taka O, Wanachiwanawin W. 51. Rahman MM, Khan L, Islam MS, Islam MM, Saha Development of flow cytometry for detection and D. A hemophilic patient presented with an abdo- quantitation of red cell bound immunoglobulin G minal mass, fever and cough. Bangabandhu Sheikh in autoimmune hemolytic anemia with negative Mujib Med Univ J. 2018; 11: 75-81. direct . Asian Pac J Allergy Immunol. 52. Ballas S, Lewis C, Noone A. Clinical, hematological 2011; 29: 364-67. and biochemical features of Hb SC disease. An J 36. Salama A. Treatment options for primary auto- Hematol. 1982; 13: 37. immune hemolytic anemia: A short comprehensive 53. Salsabil MA, Islam M, Jahan D, Khan MA. review. Transfus Med Hemother. 2015; 42: 294-301. Inherited hemoglobin disorders among apparently 37. Kwaan HC. Infection and anemia. Infect Disord healthy individuals: An analysis of 105 cases. Drug Targets. 2011; 11: 40-44. JAFMC. 2014; 10: 90-94. 38. Nicolas G, Viatte L, Bennoun M, Beaumont C, Kahn 54. Davis J, Vichinsky E, Lubin B. Current treatment of A, Vaulont S. Hepcidin, a new iron regulatory . Pediatric 1980; 10: 1. peptide. Blood Cells Mol Dis. 2002; 29: 327-35. 55. Reena Das. Disorders of haemoglobin structure and 39. Ganz T. Hepcidin: A regulator of intestinal iron synthesis. In: de Gruchy’s Clinical hematology in absorption and iron recycling by macrophages. Best medical practice. Saxena R, Pati PH, Mahapatra M th Pract Res. 2005; 18: 171-82. (eds). 6 ed. Wiley India Pvt. Ltd. India, 2013 pp 142-43. 40. Fleming MD. The regulation of hepcidin and its effects on systemic and cellular iron metabolism. 56. Higgs DR. Molecular mechanisms of α–thalasse- Hematology 2008; 151-58. mia. In: Disorders of hemoglobin: Genetics, patho- physiology and clinical management. Steinberg 41. Park CH, Valore EV, Waring AJ, Ganz T. Hepcidin, MH, Forget BG, Higgs DR, Nagel RL (eds). Cam- a urinary antimicrobial peptide synthesized in the bridge, United Kingdom. Cambridge University BSMMU J 2018; 11: 246-255 255

Press, 2001, pp 405-30. 431-69.

57. Higgs DR, Bowden DK. Clinical and laboratory 58. Weatherall DJ, Clegg JB. Inherited hemoglobin features of the α–thalassemia syndromes. In: Dis- disorders: An increasing global health problem. orders of hemoglobin; Genetics, pathophysiology Bull World Health Organ. 2001; 79: 704-12. and clinical management. Steinberg MH, Forget 59. Fucharoen S, Winichagoon P. Hemoglobinopathies BG, Higgs DR, Nagel RL (eds). Cambridge, United in Southeast Asia. Indian J Med Res. 2011; 134: 498- Kingdom. Cambridge University Press, 2001, pp 506.