A 10 month old girl with fever and cough

This article has no abstract. The first 100 words appear below:A 10 month old baby girl, hailing from the Cumilla (a city in the Chattogram Division), Bangladesh was brought to the Department of Hematology, Armed Forces Institute of Pathology, Dhaka Cantonment, Dhaka, Bangladesh with the complaints of fever and cough for the last seven days. With these complaints, the parents initially took the child to a local child specialist. The child specialist gave initial supportive medications and advised to investigate the complete blood count, urine routine and microscopic examination and Widal test. After 2 days, the parents again took the baby to the child specialist for reviewing the child and to show the investigations result.

After 2 days, the parents again took the baby to the child specialist for reviewing the child and to show the investigations result.The child and all investigation's results were reviewed by the child specialist and again advised to do hemoglobin electrophoresis.The parents did the hemoglobin electrophoresis and showed it to the child specialist.After reviewing the hemoglobin electrophoresis, the child specialist referred the case to a pediatric hematologist for further management.
With the medications prescribed by the child specialist of Cumilla, the child got cured but as the child repeatedly got sick after neonatal period, so the parents with anxiety and tension took the child to a pediatric hematologist in Dhaka for better management and treatment.
Pediatric hematologist requested the parents to repeat complete blood count and hemoglobin electrophoresis.Therefore, the parents did these tests in the Armed Forces Institute of Pathology where the concerned hematologist interviewed the parents and examine the baby.On general examination, mild anemia was found, her milestones of development were age proportional, jaundice, clubbing, koilonychia, angular stomatitis were absent and there were no bleeding manifestations.All other parameters were within normal limit.Systemic examinations revealed no abnormality as well as lymphadenopathy and hepatosplenomegaly were absent.Vaccination was done as per the national immunization schedule.The baby was delivered per vaginally normally and she is the only child of the non-consanguineous parents.
Feeding history is also good and taking all types of solid and liquid foods.The parents are also apparently healthy, the father is working in an industry and mother is a housewife.Her maternal and paternal uncles and aunts are also apparently healthy.With such findings, I made a provisional diagnosis and for proper diagnosis, I advised to repeat complete blood count, peripheral blood film examination, hemoglobin electrophoresis and reticulocyte count.

Differential Diagnosis
Dr. Mohammad Mizanur Rahman: While constructing the differential diagnosis, I considered the age of the child, recurrent mild illness during the neonatal period and childhood and socio-economic status.As there was no abnormality other than the mild anemia, the following differential diagnoses were considered.

Iron deficiency anemia
Among the global health problems, iron deficiency and iron deficiency anemia are the leading issues encountered in everyday clinical practice.The prevalence of iron deficiency anemia is decreasing gradually but still, it is the top-ranking cause of anemia worldwide and has the significant sequel on the lives of young children and post-menopausal women in both low-income and developed countries. 1Iron deficiency anemia may occur at any age but in infancy and childhood, it is one of the most common forms of anemia.The greatest prevalence is in between the ages of 6 and 24 months but may occur up to the age of 5 years.Iron deficiency anemia is rather infrequent in school -going children. 2 Iron is an essential and pivotal element that is required in several biologic functions such as respiration, energy production, DNA synthesis and cell proliferation. 3uman body has developed to preserve the iron in a number of ways such as the recycling of iron after the breakdown of red cells and retention of iron in the absence of an excretion mechanism.The physiology of iron homeostasis is now well-understood and involves a number of proteins including duodenal ferric reductase, divalent metal transporter 1, ferroportin, hephaestin, transferrin receptor 1 and 2, hemochromatosis factor and hemojuvelin.All such proteins are expressed at highest levels in the first part of the duodenum as well as pH and redox potential of this part of the intestine favors iron absorption.For the absorption of non-heme and heme iron, at least three pathways are known.Absorption of non-heme iron utilizes divalent metal transporter 1 (ferrous valence) and/or mobilferrinintegrin-paraferritin [ferric valence] and heme iron is absorbed through the third pathway utilizing heme oxygenase.Iron absorption depends on the body's need for iron.Active erythropoiesis and/or iron deficiency increases iron absorption but on the other hand, iron overload down-regulates its absorption.Iron absorption involves two notable steps: Mucosal uptake of iron and transfer of this iron from mucosal cell to the lamina propria where it enters into the plasma.Specific iron carrier proteins are required in both steps. 2 Excess free iron are toxic to the human body and participate in Fenton reaction to generate free radicals that cause oxidative cell damage, that's why its absorption is limited to 1-2 mg daily.
The daily iron requirement is about 25 mg and most are provided through recycling by macrophages that phagocytes senescent erythrocytes. 4, 5Both iron absorption and release from the body stores are tightly controlled by a hormone, hepcidin. 3Hepcidin is a hormone synthesized by the liver and plays a vital role in iron homeostasis.It also acts as an acute phase protein that acclimatizes the variation 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 macrophages and iron transport across the placenta. 6aised plasma hepcidin level is found in hemochromatosis, 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. 7ring the third trimester of pregnancy, 80% of the iron present in the newborn term infant is accumulated from the mother.Therefore, premature infants 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. 8The Institute of Medicine 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. 9Anemia is labeled for both male and female children aged   12 Maternal iron crossing through the placenta is the sole source of iron for the fetus in utero.Full-term fetus contains iron in about 75 mg/kg body weight in the last trimester of pregnancy and such iron stores are adequate enough for erythropoiesis in the initial 6 months of life.In children, the etiologies of iron deficiency anemia are insufficient intake with rapid growth, low birth weight and gastrointestinal losses due to exorbitant consumption of cow's milk.In low birth weight infants, the iron stores are exhausted earlier as they are smaller.In such cases, the risk of iron deficiency anemia can be reduced by delayed clumping of the umbilical cord after delivery. 13Iron content in breast milk is highest in the first month and then reduces gradually in the ensuing periods and reaches to 0.3 mg/L in about fifth month. 14However, iron content in breast milk cannot be increased by providing iron-rich foods to the mother but breast milk's iron absorption is high (50%) in comparison to food iron.Also, absorption of iron in breast milk is reduced if given with other foods to infants.Therefore, though absorption of iron in breast milk is high in comparison to the growth of the child, it is inadequate.During the first 6 months, infants use the iron from their iron stores and then additional iron is required from foods for normal erythropoiesis in the subsequent period. 15olid foods rich in iron should be given after 6 months to maintain normal erythropoiesis.Foods such as meat, fish, eggs and green leafy vegetables are rich in iron and such foods should be given to infants after the age of 6 months to meet the iron requirement.Excessive consumption of cow's milk by the infants may cause chronic blood loss due to heat-sensitive protein present in the cow's milk and presence of calcium as well as casein phosphopeptides in cow's milk impairs iron absorption resulting in iron deficiency anemia. 12However, a commonly accepted generality is that dietary deficiency alone is not sufficient to cause a clinically significant iron deficiency, therefore attempt should always be made to find out a source of blood loss as a cause of iron deficiency after the age of 1 year. 16n general, anemia produces non-specific pallor of the mucous membrane.Specific to iron deficiency anemia are epithelial tissue abnormalities such as esophageal webbing, koilonychias, glossitis, angular stomatitis and gastric atrophy.The relationship between iron deficiency anemia and such epithelial changes is not clear. 17History and physical examination may elicit the etiology of iron deficiency anemia but laboratory investigations such as complete blood count, peripheral blood film study, serum iron, total iron binding capacity and serum ferritin are usually sufficient to establish an uncomplicated iron deficiency anemia.Evaluation of hemoglobin electrophoresis, measurement of hemoglobin A2, red cell distribution width (RDW) and hemoglobin content of reticulocyte are helpful to differentiate from other microcytic anemia. 18Age of the patient and presence of anemia favors the diagnosis of iron deficiency anemia in this child but age proportional development, the absence of features such as angular stomatitis, koilonychia and history of taking iron-rich foods are the points against the diagnosis of iron deficiency anemia.

Acquired hemolytic anemia
Increased red cell destruction may result from either inherited or acquired disorders.Hemolytic anemia results when compensatory increased erythropoiesis by the bone marrow is unable to recompense increased red cell destruction.The common causes of acquired hemolytic anemia from infancy to toddlerhood are: Antibody-mediated hemolysis, hemolytic uremic syndrome, druginduced hemolysis and disseminated intravascular hemolysis. 19Antibody-mediated hemolysis is the most important etiology of acquired hemolytic anemia.Two types of antibodies such as autoantibodies and alloantibodies are usually responsible for the development of antibody-mediated hemolytic anemia.Autoantibodies are produced by the patient's own immune system and directed against epitopes of the patient's own red cell antigens. 20Characteristic features of the autoantibody determine the type of AIHA.Ig class of the autoantibody and thermal features i.e., the optimal temperature of the reaction at which autoantibody reacts with autologous red cells divided AIHA into three types such as warm AIHA, cold hemagglutinin disease (CHAD) and mixed forms. 21AIHA may also be classified as primary (idiopathic), in which red cell destruction occurs in the absence of any other coexisting disorder, or as a secondary form in which hemolysis occurs secondary to an underlying disease.Warm AIHA is usually idiopathic or secondary to other autoimmune disorders.Acute CHAD are mainly secondary to infections while chronic CHAD is predominantly associated with lymphoproliferative or neoplastic diseases. 22Genetic predisposition and environmental factors are important in the pathogenesis of autoimmunity.Several autoimmune diseases have HLA association but still no HLA linkage has been identified in AIHA.Mechanisms involving the breakdown of central and peripheral tolerance in the pathogenesis of AIHA are substantially unknown. 23Another mechanism of autoimmunity is molecular mimicry involving self-antigens and foreign pathogens.Most probably this mechanism is mainly responsible in the pathogenesis of AIHA secondary to various bacterial, mycoplasma or virus infections likely due to the presence of common antigenic epitopes on red cell proteins or carbohydrates.Polyclonal lymphocyte activation caused by several viral infections and the emergence of "forbidden clones" in lymphoproliferative disorders are more other mechanisms of development of AIHA. 24Another form of AIHA is drug-induced AIHA where environmental agents or drugs take part in the development of neoantigens which stimulate antibody production against red cells culminating the destruction of RBCs until the autoimmune response is destroyed and removed from the body. 25Autoantibodies against red cells have also been detected in about 30% of individuals who previously received red cell transfusion for any reason where transfused red cells elicit the generation of autoantibodies. 26Many other hypotheses about autoimmune/inflammatory diseases have come to the attention in the recent years but the most agreed mechanism is the loss of supperssor mechanisms and breakdown or alteration of self-tolerance. 23G is the most common antibody formed against red cells in AIHA and causes extravascular hemolysis through antibody-dependent cell mediated cytotoxicity (ADCC) in the reticuloendothelial system especially in the spleen.Red cell breakdown is influenced by the subclass of IgG; IgG1 together with IgG3 shortens the half-life more effectively than IgG2 and IgG4.These autoantibodies are directed against the epitopes of Rh system and react at 37°C, liable for the warm type of AIHA.Occasionally IgA auto-antibodies are generated and usually associated with IgG, rarely operative as the single etiology of AIHA. 27IgM autoantibodies are also formed in this disease causing mainly intravascular hemolysis as these antibodies are pentameric, capable of fixing complement more efficiently but extravascular hemolysis mainly in the liver occurs when it is C3d mediated.Autoantibodies of these type are directed against I/i blood group system and react optimally at 4°C, categorizing it as a cold form of AIHA.Nonetheless, the range of the thermal amplitude of IgM autoantibodies is 0-34°C.
If the thermal amplitude of IgM autoantibodies is close to physiological temperature (Warm IgM), then the severe form of AIHA ensures in which cases the mortality rate may be 18-25%. 25Intravascular hemolysis in AIHA is more detrimental and severe than extravascular hemolysis as the rate of RBC destruction is 200 mL per hour in case of intravascular hemolysis which is 10 times more than extravascular hemolysis. 22HA may occur after allogeneic hemopoietic stem cell transplantation.The 3 year cumulative incidence in such a population is about 4.4%. 28, 29AIHA is rare with an estimated incidence of 0.2 per million individuals below 20 years of age. 30Peak incidence is found in less than four years of age. 31The mortality rate is approximately 11%. 32Most patients with AIHA presents their clinical manifestation insidiously but some patients have severe acute symptoms.Symptoms may range from asymptomatic compensated reticulocytosis with mild hyperbilirubinemia to acute fulminant hemolysis leading to jaundice, hepatosplenomegaly, tachycardia and angina.They are also determined by the causative factor responsible for AIHA and degree of hemolysis which again depends on the type of autoantibody.IgM warm reactive AIHA presents with life-threatening acute intravascular hemolysis with high mortality rate compared to patients with cold type AIHA where the symptoms are usually mild. 33Bone marrow compensatory ability also determine the degree and severity of anemia.Those patients with AIHA who respond their anemia by reticulocytosis usually presents with less severe clinical symptoms than patients who do not respond by reticulocytosis rather develop reticulocytopenia.Such reticulocytopenia are observed in 20% of adult and 38% of children with AIHA. 34 On the other hand, due to the presence of coexistence morbid conditions such as diabetes, uncontrolled hypertension, obesity, osteoporosis, peptic ulcer or children where treatment with corticosteroid is relatively contraindicated and in such cases second-line therapy i.e., splenectomy is thought to be the most effective and produce high response rates up to 82%.Rituximab (so-called "medical splenectomy") is another effective secondline therapy, providing high initial response rates up to 85% and prolonged disease-free survivals of 55% up to 2 years with reduced adverse reactions. 36n this patient, age, presence of anemia and possible viral respiratory tract infections favors the diagnosis of AIHA but chronic as well as less severe anemia for a longer duration are the points against the diagnosis of AIHA.

Concurrent infections
Anemia is one of the most noticeable features of infection.The severity of anemia in infection ranges from a mild, asymptomatic form to an acute lifethreatening complication of infection.Anemia of chronic disease is defined as the anemia seen in patients with chronic infections.In addition to infections, anemia is also seen in other chronic diseases including cancer and chronic inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease and renal failure.This anemia is referred to as anemia of inflammation.In acute inflammation, anemia is directly related to bone marrow suppression and acute hemolysis but in chronic inflammation, several factors are involved in the pathogenesis of anemia.Inflammatory cytokines, hepcidin and bacterial toxins play a signi-ficant role.The anemia in chronic inflammation is usually normocytic normochromic and refractory to therapy with hematinics. 37][40] Hepcidin is synthesized in the liver, present in plasma and excreted through urine and functions as a major regulatory hormone for iron transport. 41epcidin controls the activity of ferroportin 1, a transmembrane domain protein present on the surface of enterocytes, hepatocytes and macrphages.The movement of iron through these cells' wall is regulated by ferroportein 1.By binding with ferroportein 1, hepcidin inhibits the function of ferroportein 1 and thereby prevents the transportation and exit of iron through the cell membrane.As a consequence, mobilization from hepatocytes and release of sequestered iron from macrophages is impaired and result in a decrease in the availability of iron for erythropoiesis.In inflammatory states, hepcidin synthesis is increased mainly by the cytokine IL-6 and thus preventing the body from more iron absorption and inhibiting iron transportation.Hence, the response of anemia of inflammation to EPO and EPO stimulating agents is suboptimal. 42Anemia of inflammation is an anemia that develops within 1-2 months of the onset of illness.Anemia persists without increasing in severity.4][45] The anemia is usually mild and morphologically either normocytic normochromic or microcytic hypochromic anemia.The clinical features are those of the underlying disorders. 46nemia of inflammation is diagnosed by the presence of anemia in association with inflammatory or infectious disease.Laboratory investigations revealed low serum iron, decreased soluble transferrin receptors and reduced transferrin saturation with iron.Reticuloendothelial iron stores are increased depicted by Perl stain of bone marrow sample and increased serum ferritin. 47Anemia of inflammation is usually treated by alleviating the underlying disorders.Iron therapy is unsatisfactory unless there is a concomitant iron deficiency.Administration of EPO may provide mild or suboptimal response. 37is girl has been suffering from upper respiratory tract infection and fever which favor the diagnosis of anemia of inflammation but presence of weakness from toddlerhood; less playful and persistent mild anemia are the points against the diagnosis of anemia of inflammation.

Congenital hemolytic anemia
Premature destruction of red cells is called hemolysis.Mild hemolysis is not manifested as anemia but if the erythrocyte loss is more than the compensatory activity of the bone marrow then only anemia develops which may be of varying severity. 48There are a wide range of causes, both congenital and acquired, responsible for hemolysis. 49, 50Red cell membrane, enzyme and hemoglobin defects are the main etiologies of congenital hemolytic anemia.In congenital hemolytic anemia the genes that master the production of red cells are defective.On the basis of the affected defective genes, different types of congenital hemolytic anemia develop.In each type, abnormal erythrocytes are produced in the body and destroyed prematurely. 51Hemolytic anemia in children should be suspected in any child with anemia, jaundice and splenomegaly.In severe cases, skeletal deformities especially of the skull and facial bones due to erythroid hyperplasia and cardiomegaly due to high cardiac out may be demonstrated by radiographs.A few patients may also develop pigment gallstones after the first decade of life.
Children with mild hemolytic anemia may have nearly normal hemoglobin levels if reticulocyte production increases sufficiently for the increased rate of RBC destruction.However, concurrent infectious illness may aggravate the anemia during which mild hemolytic cases can be identified.Hemoglobin level falls during infectious illness due to oxidative stress and depression of bone marrow activity. 52Examination of blood film is the mainstay in the evaluation of hemolytic anemia and in some cases, the morphology of red cells may point to the diagnosis of a typical congenital hemolytic anemia.8 In Hb Bart's hydrops fetalis, its amount is approximately 80-85% which is incompatible with life. 55Here, I like to mention that there is no conclusive data regarding the amount of Hb Bart's when it is in between 25-80%, then what should be nature of the condition.Therefore we recommend studying and analyzing alpha thalassemia gene in more cases to resolve the queries when the amount of Hb Bart's is in between 25 to 80% as no conclusive data is available in the Dr. Debashish Saha: What type of advice should be given to the parents?
Dr. Rahman: Genetic counseling is most important advice in such inherited disorders as such counseling reduces the reproductive risk of the family.

Figure 1 :
Figure 1: Hemoglobin patterns of the patient (A) and father (B)

Figure 2 :
Figure 2: Pedigree chart of the patient

Table I Hematological parameters of the patient and her parents
binding capacity.Iron deficiency anemia is the most common nutritional deficiency anemia in infancy and in adolescents who have menstruation but those having increased growth rate and cannot meet their requirements are at increased risk of developing iron deficiency anemia. 2, 57en two α-globin genes deleted or inactivated, either both on the same chromosome 16 (in cis) or on one each of the two chromosomes 16 (in trans), the affected person is well but has borderline anemia, as well as microcytic hypochromic red blood cells.57LowHbA2level is found mainly in iron deficiency anemia and alpha thalassemia.Delta beta thalassemia and hereditary persistence of fetal hemoglobin are other conditions which may cause low HbA2.Dr.Imana: Did you exclude iron deficiency anemia in this case as low HbA2 indicate the possibility of iron deficiency anemia?Dr.Monwar Tark: In this patient blood film showed no specific features such as elongated and pencilshaped cells which are characteristic of iron deficiency anemia.Moreover, serum iron profile was done and it was within the reference limit.Dr.Durdana Mahin: What is the prognosis of this patient and how will follow up this case?Dr.Rahman: The patient will remain asymptomatic unless affected by any conditions which suppress the bone marrow.She should be followed-up by doing hemoglobin level monthly and serum ferritin six monthly.Dr.Arif Ahmed Khan: How will you confirm the diagnosis and have you done it?Dr.Moin: By doing alpha thalassemia mutation study, we can confirm the diagnosis.However, family screening and pedigree study (Figure2) showed the presence of Hb Bart's in her father and these findings almost confirm the diagnosis of either alpha thalassemia carrier or trait.I have not done the molecular diagnosis as it is not available in our country at present.Dr.Elias: By quantifying Hb Bart's by capillary hemoglobin electrophoresis, how can we diagnose different types of alpha thalassemia?Dr. Rahman: Presence of Hb Bart's less than 25% indicates the presumptive diagnosis of either alpha thalassemia carrier or alpha thalassemia trait.Hb Bart's ≥ 25% indicates the diagnosis of HbH disease in neonates.
59Alpha thalassemia is caused by mutations or deletions affecting either one or more α-globin genes, leading to decreased or absent α-globin chain production from the affected gene(s).59Dr. Luhar Khan: What is the cut off value of Hb Bart's for diagnosing alpha thalassemia trait or carrier?Dr.Moin: Two types of information regarding the quantity of Hb Bart's are available.However, if the Hb Bart's level is less than 25%, the condition is likely to be either alpha thalassemia carrier or alpha thalassemia trait.Here I want to mention that in most published literates such cut off value of Hb Bart's is given for neonates but here we found such amount of Hb Bart's both in the adult patient and his 10-month old daughter.Dr. Shourov: Here the HbA2 level is low.What are the conditions where HbA2 level is low?Dr. Monirul Islam: Dr. Zannat: Did low HbA2 level in this case support the diagnosis?Dr. Shahidul Islam: In this case low HbA2 level along with the presence of moderate amount of Hb Bart's strongly suggest the presence of either alpha thalassemia carrier or alpha thalassemia trait.