Benign Hematologic Disorders in Children: A Clinical Guide

This book provides a comprehensive overview of benign hematologic disorders in children. Divided into nine sections, the text reviews common hematologic disorders or conditions that affect children, while providing state-of-the-art information on pathophysiology, diagnosis, treatment, and management strategies.

The text begins with a section on hematopoiesis, and the next section covers red blood cell disorders. The following sections provide overviews of platelet disorders, white blood cell disorders, and coagulation disorders. The sixth and seventh sections discuss neonatal hematology and bone marrow failure syndrome. The eighth section reviews supportive care, while the final section covers miscellaneous subjects including pediatric vascular anomalies and complement dysregulation syndromes.
Written by experts in the field, Benign Hematologic Disorders in Children: A Clinical Guide is a valuable resourcefor clinicians and practitioners who treat children afflicted with these disorders.

• Language: English
• Publisher: Springer
• Release date: Aug 18, 2020
• ISBN: 9783030499808

Book preview

Part IOrigin of Blood Cells

© Springer Nature Switzerland AG 2021

D. M. Kamat, M. Frei-Jones (eds.)Benign Hematologic Disorders in Childrenhttps://doi.org/10.1007/978-3-030-49980-8_1

1. Hematopoiesis

Chintan Parekh¹  

(1)

Children’s Hospital Los Angeles and the Keck School of Medicine at the University of Southern California, Los Angeles, CA, USA

Chintan Parekh

Email: [e-mail]

Keywords

Hematopoietic differentiationTranscription factorsCytokinesBone marrow microenvironment

Introduction

Blood cell development (hematopoiesis) represents one of the most well-studied tissue development processes. The use of flow cytometry to define hematopoietic cell types, the investigation of hematopoietic cell differentiation pathways in cell culture and mouse models, and the characterization of molecular mechanisms through gene expression profiling and genetic manipulation experiments in human cells and mouse models have yielded a high-resolution picture of the cellular and molecular processes underlying hematopoiesis. A knowledge of the fundamentals of normal hematopoiesis is critical for understanding the pathophysiology, diagnosis, and management of blood disorders. Furthermore, the advent of next-generation sequencing -based molecular testing for diagnosis, prognostication, and management of hematological diseases in clinical practice makes a basic knowledge of the molecular mechanisms driving hematopoiesis an essential part of the clinician’s toolkit. This chapter provides an overview of the biology of normal hematopoiesis from the perspective of clinically applicable aspects of blood cell development.

Cell Development Stages during Hematopoiesis

All blood cells are derived from self-renewing hematopoietic stem cells (HSC), which in postnatal life reside in the bone marrow. The presence of HSC in the bone marrow that can give rise to the entire blood cell system has been shown in both laboratory experiments in mice and in patients receiving bone marrow transplantation. HSC give rise to different blood cell lineages through differentiation into progenitor cell types that lack self-renewal capacity and are progressively more restricted in their lineage potential [1].

Progenitor cell types with distinct lineage potentials have been defined by specific cell surface antigen expression profiles using flow cytometry [2]. Lineage readouts from in vitro culture assays and the transplantation of human hematopoietic cells into immunodeficient mice (xenotransplantation) have formed the mainstay for defining the functional properties of these progenitor cell types [3]. Hematopoietic stem and progenitor cells (HSPC) are characterized by the expression of CD34. In the classical model of hematopoiesis (Fig. 1.1), the origin of common myeloid (CMP) and lymphoid progenitors (CLP) represents the bifurcation of myeloid and lymphoid lineages. Further lineage separation downstream of CMP occurs through the generation of megakaryocytic-erythroid (MEP) and granulocytic-monocytic (GMP) progenitors [1]. While the classical model is based on strict dichotomy of lineages, recent single-cell studies indicate that lineage separation may be a more continuous process where lineage biases are established in HSC and early progenitors and alternative lineage potentials are gradually extinguished to give rise to unilineage progenitors [15]. The abovementioned newer model proposes the direct origin of unilineage progenitors (e.g., megakaryocytic or erythroid progenitors) from multilineage ancestors (HSC, multipotent progenitors, CMP) rather than through an intermediate bilineage progenitor like the MEP [1]. Clinical observations of abnormalities in more than one lineage in disorders that primarily affect a single lineage are consistent with a close developmental relationship between certain lineages. For instance, iron deficiency anemia is characterized by thrombocytosis, an observation consistent with the existence of MEP [16].

../images/483850_1_En_1_Chapter/483850_1_En_1_Fig1_HTML.png

Fig. 1.1

Classical model of hematopoiesis. HSC hematopoietic stem cell, MPP multipotent progenitor, LMPP lymphoid-primed multipotent progenitor, CMP common myeloid progenitor, GMP granulocytic-monocytic progenitor, MEP megakaryocytic-erythroid progenitor, MDP monocyte-dendritic progenitor, meg megakaryocyte. A subset of the key transcription factors [4–12] (red) and cytokines [13, 14] (blue) for HSPC cell types and lineages are depicted

HSC are quiescent cells that divide infrequently, approximately once every 40 weeks [17], a feature that protects these long-lived cells from the accumulation of leukemogenic mutations from DNA damage due to environmental agents. In contrast, downstream progenitor cells are highly proliferative and underlie the cell number amplification required to produce the millions of blood cells that need to be generated every day. Differentiation of unilineage progenitors is associated with a loss of proliferative capacity, and terminally differentiated erythroid and myeloid progeny are nondividing cells. The rapidly dividing progenitor cells are particularly sensitive to apoptosis from DNA damage, a phenomenon that accounts for cytopenias seen after treatment with many chemotherapeutic agents. Recovery from post-chemotherapy cytopenia is driven by the activation of quiescent HSC , which are much more resistant to such DNA damage [18].

Sites of Hematopoiesis

During embryonic development in humans, hematopoiesis is first seen in the yolk sac at 3 weeks of gestational age. However, this primitive hematopoiesis is limited to the generation of red cells, macrophages, and megakaryocytes [19]. The first HSC with multilineage myeloid and lymphoid potentials appears in the aorto-gonad-mesonephros and yolk sac regions at 5–6 weeks of gestational age [20]. The fetal liver is the predominant site of hematopoiesis during the second trimester [21]. The spleen forms a minor site of hematopoiesis during fetal life [22]. Liver hematopoiesis decreases in the third trimester, and the bone marrow becomes the dominant site of hematopoiesis by birth [21]. Liver hematopoiesis ceases soon after birth (usually by 5 weeks) leaving the bone marrow as the only site of physiological hematopoiesis during postnatal life [22]. While most differentiated blood cell types in postnatal life are produced in the bone marrow, T-cell production occurs in the thymus through differentiation of hematopoietic progenitors that have migrated from the bone marrow [5].

Hematopoiesis in the liver and spleen resulting in organomegaly is seen after birth (nonphysiological extramedullary hematopoiesis) in disorders with ineffective bone marrow hematopoiesis like thalassemia or as a compensatory mechanism for increased red cell destruction as in sickle cell anemia [22, 23]. While many of the underlying molecular mechanisms are shared between fetal liver and postnatal bone marrow hematopoiesis, several regulatory differences exist between the two ontogenetic phases. For instance, the switch from fetal liver to bone marrow hematopoiesis is thought to underlie the spontaneous resolution of transient myeloproliferative disorder in infants with Down syndrome, a disorder characterized by abnormal proliferation of fetal liver megakaryocytic progenitors harboring a mutation in the transcription factor gene GATA1 [24].

Mechanisms Underlying Hematopoiesis

The orderly differentiation of HSC into a myriad of blood cell types is tightly regulated by transcription factor (TF) genes in the hematopoietic cells as well as extrinsic signals from the bone marrow microenvironment including cytokines and adhesion molecule-mediated cell-to-cell interactions.

Transcription Factors

Transcription factors (TF) are DNA-binding proteins that recognize specific DNA sequences to regulate the expression of a multitude of genes. TF typically recruit histone-modifying proteins and other cooperating TF to specific DNA-binding sites to form regulatory complexes that promote or inhibit the expression of target genes [25–27]. The differentiation and self-renewal of HSC are regulated by the orderly and stage-specific expression of TF, which in turn drive the expression of stem cell, progenitor cell, or lineage-specific gene networks [4].

While some TF such as the B-cell TF PAX5 are expressed in a highly lineage-specific fashion, TF like SPI1 (myeloid and B-cell) are shared between lineages [4]. HOXB3, HOXB4, and HOXA9 TFs are highly expressed in HSC [9]. CMP show high expression of TAL1, GATA1, and CEBPA [4]. On the other hand, generation of early lymphoid-primed progenitors is associated with downregulation of TAL1 [28]. Divergence of MEP from GMP is driven by the upregulation of TAL1 and GATA1 in MEP and that of CEBPA in GMP [4]. GATA2 regulates HSC function as well as monocyte differentiation [6].

Molecular studies have revealed germline mutations in TFs as the etiology for several hematopoietic disorders (Table 1.1). GATA2 mutations result in a monocyte immunodeficiency syndrome characterized by recurrent infections [6]. RUNX1 mutations are associated with a familial thrombocytopenia and acute myeloid leukemia/myelodysplasia predisposition syndrome [29]. Germline mutations in PAX5 result in a predisposition to B-cell acute lymphoblastic leukemia [30]. While most cases of Diamond-Blackfan anemia (DBA) are caused by mutations in ribosomal genes, germline GATA1 mutations that impair the synthesis of the full-length GATA1 isoform have been reported in patients with DBA who did not have known DBA ribosomal gene anomalies [33]. The variable penetrance and age of onset of clinical disease in some of these genetic disorders have important implications for screening donors when using familial donors for hematopoietic stem cell transplantation (HSCT) for bone marrow failure and immunodeficiency syndromes [35].

Table 1.1

Hematopoietic disorders due to loss of function germline mutations in TF genes [6, 8, 10, 29–34]

Cytokines

Several growth factors or cytokines play a critical role in hematopoiesis. Cytokines activate specific receptors expressed on hematopoietic cells to regulate cell proliferation, survival, and differentiation. Lineage or differentiation stage-specific expression of cytokine receptors enables the regulation of specific blood cell types by a given cytokine. Cytokines are produced by local microenvironmental cells in the bone marrow (stem cell factor, SCF) as well as the liver (thrombopoietin) and kidney (erythropoietin). Thrombopoietin is required for the maintenance of self-renewing HSC and megakaryocytic differentiation to produce platelets. Interleukin-7 (IL-7) plays a critical role in lymphoid differentiation. G-CSF and GM-CSF are essential for myeloid differentiation into neutrophils and monocytes. Erythropoietin drives lineage commitment, survival, and differentiation of erythroid progenitors [13].

The binding of a cytokine to its receptor typically sets off a cascade of biochemical signaling events involving the phosphorylation-mediated activation of a multitude of downstream kinase proteins that ultimately lead to the transcriptional regulation of self-renewal, proliferation, cell survival, and differentiation gene networks. The JAK/STAT family of kinases, which associate with the intracellular domain of cytokine receptors, constitutes a key signal transduction pathway for several cytokines (e.g., erythropoietin, thrombopoietin) in hematopoiesis [36] (Fig. 1.2).

../images/483850_1_En_1_Chapter/483850_1_En_1_Fig2_HTML.png

Fig. 1.2

Cytokine receptor signaling in hematopoietic cells. Signaling transduction downstream of activation of the thrombopoietin receptor, MPL is depicted. P phosphorylation, JAK Janus kinase, STAT signal transducer and activator of transcription proteins

Mutations in cytokine, cytokine receptor, or signaling transduction genes account for several very rare hematopoietic disorders (Table 1.2). While these mutations account for only an exceedingly small minority of cytopenia diseases due to defective hematopoiesis, defining these germline aberrations is essential for the elucidation of the molecular mechanisms driving human hematopoiesis, the selection of appropriate treatment, and the development of new gene therapy approaches. For instance, loss of function mutations in the thrombopoietin and erythropoietin genes causes thrombocytopenia and anemia syndromes, respectively, that are clinically similar to other inherited bone marrow failure cytopenia disorders. However, unlike most bone marrow failure syndromes resulting from intrinsic defects in hematopoietic cells, patients with germline TPO or EPO mutations do not respond to HSCT and require cytokine replacement therapy (recombinant erythropoietin or thrombopoietin mimetic agents) instead. Mutations in the thrombopoietin receptor gene MPL are seen in amegakaryocytic thrombocytopenia, a disorder whose natural history is a progression to aplastic anemia. G-CSF receptor mutations have been reported in severe congenital neutropenia [14]. Mutations in the IL-7 receptor or the gamma chain component of the IL-2 receptor result in severe combined immunodeficiency due to lymphocyte defects [37].

Table 1.2

Hematopoietic disorders due to germline mutations in cytokine pathway genes [14, 37, 38]

Cell-to-Cell Interactions

HSPC reside in complex bone marrow microenvironmental niches made of osteoblast, stromal, endothelial, mesenchymal stem, and/or megakaryocyte cells [39, 40]. Several elegant studies in murine cell type-specific gene knockout models and in vitro culture systems of human hematopoietic cells have revealed the effects of specific cell populations in these niches on HSPC fate. The fate of HSC with respect to self-renewal, proliferation, and differentiation is regulated by their anatomic localization to specific niches in the bone marrow. Signals from osteoblasts in endosteal niches promote HSC self-renewal, whereas microenvironmental cues in perivascular niches promote HSC differentiation [41]. Cross talk between hematopoietic cells and the microenvironment is mediated by adhesion molecule receptors on the cell surface that interact with the extracellular matrix and adhesion molecules expressed on other cells. Specific interactions between adhesion molecule receptors and their ligands dictate the localization of HSPC to specific microenvironmental niches and induce downstream cellular proliferation, survival, self-renewal, and differentiation signaling pathways [42].

Adhesion molecule receptors expressed on HSPC, many of which overlap with those mediating the migration of leukocytes into peripheral tissues, include integrins, selectins, CXCR4, and NOTCH pathway receptors [42, 43]. CXCR4 interacts with its ligand CXCL12 expressed by microenvironmental cells including osteoblasts to promote bone marrow residence and quiescence of HSPC [44] (Fig. 1.3). Strategies to modulate adhesion molecules have been translated into therapeutic applications. For instance, CXCR4 inhibitors are clinically used to mobilize HSPC from marrow niches into the blood for collection of peripheral blood stem cells for HSCT [45].

../images/483850_1_En_1_Chapter/483850_1_En_1_Fig3_HTML.png

Fig. 1.3

Bone marrow hematopoietic microenvironment. HSPC hematopoietic stem and progenitor cells, Ost osteoblasts, Str stromal cells, Endo endothelial cells, MSC mesenchymal stem cells, Meg Megakaryocytes, ECM extracellular matrix. Bidirectional arrows indicate interactions between HSPC and the microenvironment

Conclusion

In vitro and in vivo laboratory studies of immunophenotypically defined HSPC, and the lessons learned from patients with mutations in genes critical for hematopoiesis have resulted in the elucidation of the mechanisms underlying the generation of blood cells from HSC. Mechanistic insights about the specific transcription factors and cytokines driving the hematopoietic lineage differentiation have greatly advanced the understanding of blood disorders and led to the development of molecular diagnostic tests for hematopoietic disorders.

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Part IIRed Blood Cell Disorders

© Springer Nature Switzerland AG 2021

D. M. Kamat, M. Frei-Jones (eds.)Benign Hematologic Disorders in Childrenhttps://doi.org/10.1007/978-3-030-49980-8_2

2. Nutritional Anemias: Iron Deficiency and Megaloblastic Anemia

Deanna Mitchell¹, ²  , Jessica Foley¹, ²   and Aarti Kamat³  

(1)

Helen DeVos Children’s Hospital, Grand Rapids, MI, USA

(2)

College of Human Medicine, Michigan State University, East Lansing, MI, USA

(3)

Helen DeVos Children’s Hospital Future fellow, Pediatric Hematology/Oncology – University of Michigan Medical Center, C.S. Mott Children’s Hospital, Ann Arbor, MI, USA

Deanna Mitchell (Corresponding author)

Email: [e-mail]

Jessica Foley

Email: [e-mail]

Aarti Kamat

Email: [e-mail]

Keywords

Microcytic anemiaIron deficiency anemiaMacrocytosisVitamin B12 deficiencyFolate deficiency

Iron Deficiency Anemia

Iron deficiency is the most common cause of anemia in infancy and childhood. Iron deficiency anemia has important health ramifications, as it has been associated with abnormal neurodevelopment [1]. Dietary modifications can prevent iron deficiency. If iron deficiency develops, simple interventions can help prevent cognitive detriments. Given the common prevalence of this disease and its impact on neurodevelopment, it is imperative that primary care physicians learn to recognize, diagnose, and treat this condition.

Prevalence

Iron deficiency remains the number one cause of anemia worldwide, affecting more than 2 billion people, with a considerable effect on the lives of young children, especially in low-income and developing countries [2]. The prevalence of iron deficiency anemia is lower in the USA in comparison to developing countries; however, it remains a common problem, with a prevalence of 1.6–7.4% among children less than 5 years of age. Iron deficiency affects at least 2.4 million children in the USA [3].

In the USA, iron deficiency is more common among children living at or below the poverty level. Hispanic/Latino and Asian immigrant children have significantly higher rates of iron deficiency (17%) compared to Caucasian children 6% [4]. Prevalence of iron deficiency differs depending on body mass with obese toddlers having a higher prevalence compared to normal weight toddlers (20% vs. 7%) [3]. Children not in daycare and overweight have higher odds of iron deficiency. Other factors associated with iron deficiency include premature birth and low birth weight. The rate of iron deficiency decreases after 2 years until adolescence when 8–10% of teenage girls may develop iron deficiency and 3% will be diagnosed with iron deficiency anemia [5].

Pathophysiology

Iron is critical for many body functions including energy production, respiration, DNA synthesis, and cell proliferation. Erythropoiesis, or red blood cell production, is dependent upon iron. Nearly 75% of iron is bound to heme proteins, hemoglobin, and myoglobin and is involved in oxygen transport and storage. Thirty percent of iron is otherwise bound in storage proteins: ferritin and hemosiderin of small portions are bound in critical enzyme systems, including catalase and cytochromes [6].

Total body iron increases dramatically in the first year of life which allows nearly 80% of iron to be used for hemoglobin production and iron stores. A newborn infant’s body contains 0.3–0.5 grams of iron, whereas the total iron content of an adult is 4–5 grams. An average of 0.8 mg of iron must be absorbed each day during the first 15 years of life. Iron metabolism is a relatively closed system, in which iron is continuously recycled to meet the demands of the body but particularly for red blood cell production. After full growth is achieved, the total body iron remains fixed within narrow limits. When red blood cells die, they are removed from circulation by macrophages, and iron is extracted from hemoglobin and made available to the plasma protein, transferrin, for transport to the marrow [6].

As children grow and body iron content increases, less iron is needed from the diet to meet their needs. Approximately 5% of the iron requirement for erythropoiesis comes from dietary intake in an adult, while 30% of the iron requirement for erythropoiesis must come from the diet in children [1]. Iron loss from the body is minimal and usually due to exfoliation of mucous membranes and skin. No known specific mechanisms for iron excretion through the liver or kidneys exist. Iron balance is predominantly achieved through modulation in the absorption of iron in the intestine. The average adult diet contains 10–15 mg of iron daily. Approximately 8–15 mg of iron daily is needed for optimal nutrition [6]. Dietary iron absorption in the intestine varies from 5% to 20% depending upon the physiological need. For example, intestinal absorption may increase fourfold in the setting of blood loss, pregnancy, sports activity, and hemolytic anemia [1].

Iron is absorbed mainly in the small intestine, mostly in the duodenum and first part of the jejunum. Iron absorption is regulated by the peptide hormone hepcidin, which regulates how much iron is taken up in the intestine and transported to the plasma. Hepcidin is synthesized primarily in the liver. Increased hepcidin levels decrease the intestinal absorption of iron by binding to and inducing the degradation of ferroportin, a transmembrane protein located on the surface of intestinal enterocytes, which facilitates the absorption of iron from the intestine. Hepcidin is regulated by the iron demand of the body. When the body is iron deficient, hepcidin concentrations are low, and thus ferroportin is available to absorb intestinal iron. Serum hepcidin expression, and subsequent intestinal iron absorption, is affected by iron stores in the form of transferrin and ferritin, erythropoiesis, bioavailability of dietary iron, and inflammatory states [7].

Iron Deficiency and Neurodevelopment

Iron deficiency has been strongly linked to long-term neurological complications that affect cognitive, social, and behavioral development in infants and young children [8]. Iron deficiency, even without anemia, can adversely impact the social-emotional behavior of infants and influence their relationship with their caregiver. One study noted that infants with iron deficiency demonstrated increased shyness, decreased soothability, and decreased engagement [9]. Iron deficiency has its greatest impact when it occurs during fetal growth or in the first few years of life when neural systems are developing [8]. Even after iron supplementation, the cognitive and social impairments can persist in children that were formerly iron deficient [8]. One study demonstrated children who were iron deficient as infants had slower reaction times and worse inhibitory control 8–9 years after iron therapy [10].

Adolescent girls are at risk of iron deficiency and iron deficiency anemia. In a survey of adolescents in the USA, those who were iron deficient were found to have lower math scores [11]. Another study looked at iron supplementation in adolescent girls who were not anemic but had serum ferritin ≤12 micrograms/L, corresponding to iron deficiency. The girls who received iron supplementation were noted to have an improved ferritin and performed significantly better on tests of verbal learning and memory [12]. Blood loss from menstruation is expected in adolescent girls; however the resulting iron deficiency is underdiagnosed resulting in a lack of recognition of the cognitive, social, and behavioral consequences of iron deficiency in this population. Iron deficiency during adolescence occurs at a time when education is imperative to achieve a successful adulthood, and thus girls with heavy menses may suffer neurologic sequelae that will diminish their academic potential. Iron stores should be optimized prior to childbearing years [5].

Iron Deficiency and Thrombosis and Stroke in Children

The association of thrombosis and iron deficiency in both children and adults has been described [13]. Thrombotic complications of iron deficiency anemia have been attributed to the secondary thrombocytosis which occurs in one third of patients. However, cerebral venous sinus thrombosis associated with iron deficiency has been reported without the presence of thrombocytosis [14]. Hartfield et al. described a case series of six children who developed an ischemic stroke or venous thrombosis associated with iron deficiency [15]. Iron may contribute to a hypercoagulable condition by affecting blood flow patterns within vessels due to reduced deformability and increased viscosity of microcytic red blood cells [15]. Hypoxic injury is seen particularly in the areas of the brain supplied by end arteries, such as the basal ganglia, thalamus, and hypothalamus [16]. Maguire et al. additionally described [15] previously healthy pediatric patients between 12 and 38 months of age who developed stroke and found iron deficiency anemia in 53% (8/15) of the patients. For children who were previously healthy, those who went on to develop stroke were ten times more likely to have iron deficiency than those who remained healthy and did not develop stroke. Children with iron-deficiency anemia accounted for more than half of all stroke cases in children without an underlying medical illness. This suggests that iron deficiency may be a risk factor for stroke in children [17].

Etiology of Iron Deficiency

The causes of iron deficiency in children include one or more of the following: inadequate reserves at birth, inadequate intake of iron in the diet, reduced intestinal absorption of iron, or chronic blood loss.

Many intrauterine conditions may cause decreased iron reserves at birth, including prematurity, twin gestation, intrauterine fetus-fetus and fetus-maternal transfusions, exchange transfusion at birth, severe iron deficiency anemia in the mother, and/or early clamping of the umbilical cord. Eighty percent of the iron present in the newborn term infant is accumulated during the third trimester of pregnancy. Therefore, infants who are born prematurely have lower iron stores. Other maternal conditions, including anemia, diabetes, conditions that cause intrauterine growth restriction, and maternal hypertension can contribute to low fetal iron stores in preterm and term babies. Additionally, premature infants require frequent lab monitoring during hospitalization which can result in iatrogenic anemia and may cause iron deficiency. The use of recombinant human erythropoietin in preterm infants in an attempt to prevent transfusion has been associated with further depletion of iron stores. Therefore, premature infants in particular require iron supplementation; this should be given at a dose of of 2–4 mg/kg/day to pretrerm and low-birth-weight infants before 6 months of age [1].

Healthy, term infants have iron stores of approximately 75 mg/kg and a mean hemoglobin of 15–17 g/dl. Term infants generally have sufficient iron stores for the first 4–6 months. During this time they are either receiving breast milk or iron-supplemented formula. All standard infant formulas contain a minimum of 6.7 mg/l of iron. However after 4–6 months, an infant’s diet should include iron-fortified foods such as cereals or iron-supplemented formula. Inadequate iron in infants less than 12 months of age is most commonly due to either breastfeeding without the initiation of iron supplementation or iron-rich foods by 6 months of age [1]. Occasionally, infants have non-fortified cow’s milk introduced prior to 12 months of age. Cow’s milk does not contain appropriate concentrations of iron for infants. Both human milk and cow’s milk have low concentrations of iron; however, the bioavailability of iron is much greater from human milk. Male et al. demonstrated in 488 healthy infants in 11 European countries that 7.2% were iron deficient at 12 months and 2.3% had iron deficiency anemia. Toddlers who were fed cow’s milk had a progressively increasing risk of iron deficiency rising to 39% [18]. Additionally, infants can also experience microscopic blood loss secondary to cow’s milk protein-induced colitis. This chronic blood loss contributes to the severe iron deficiency seen in excessive cow’s milk ingestion. Young children may develop a severe form of this syndrome and have significant protein-losing enteropathy with hypoalbuminemia and edema as well as iron deficiency anemia [19]. In contrast to heme iron, the absorption of nonheme iron is inhibited by casein and calcium in milk. Casein and calcium are present in much higher concentrations in cow’s milk compared to human milk [20].

Iron deficiency due to inadequate dietary intake is not just limited to children. In developing countries, iron deficiency anemia is most often secondary to insufficient iron in the diet. However, loss of blood from intestinal parasites also contributes to inadequate iron stores [2]. The most common cause of iron deficiency in adults in high-income countries is secondary to pathologic conditions such as gastrointestinal bleeding or secondary to a strict vegetarian diet. Vegetarians are at risk of iron deficiency for several reasons. A cereal-based diet decreases the iron bioavailability because phytates in grains form a complex with iron which is then poorly absorbable. Additionally, intestinal absorption of iron depends upon the form of iron ingested. Heme iron found in meat, fish, and poultry is readily absorbed with a higher bioavailability compared to nonheme iron which is found in some plants. The most readily absorbed iron is found in red meat, such as beef. Thus vegetarians should actively manage their dietary intake of iron or consider supplementation [21].

Gastrointestinal disorders including celiac disease, inflammatory bowel disease (Crohn disease), giardiasis, or other malabsorption conditions involving the duodenum can result in inadequate absorption of iron and anemia. Premature infants who suffered from necrotizing enterocolitis and have short gut syndrome frequently absorb inadequate iron [22]. Patients who have undergone bariatric surgery also are at risk for lack of iron absorption and subsequent anemia [23]. Inflammatory bowel disease, juvenile polyposis syndrome, cow’s milk protein-induced colitis, and chronic aspirin can result in increased gastrointestinal blood loss and chronic iron deficiency anemia [24].

Additional causes of iron deficiency that should be considered in a differential diagnosis include hemolysis. In rare forms of intravascular hemolysis, like paroxysmal nocturnal hemoglobinuria, iron is lost in the urine, and iron deficiency contributes to the existing hemolytic anemia. Occasionally iron deficiency is multifactorial, such as in runners and endurance athletes. Here iron deficiency may be partially due to hemolysis, blood loss, and mild inflammation [25]. There are patients with homozygous mutations in TMPRSS6, encoding the hepcidin inhibitor matriptase-2, a rare genetic form of anemia known as iron-refractory iron deficiency anemia (IRIDA). Patients with IRIDA have high serum hepcidin levels and therefore poor or no response to oral iron [26].

Screening

Screening for anemia with hemoglobin evaluation is recommended at 1 year of age by the American Academy of Pediatrics (AAP). In addition to obtaining a hemoglobin, screening for risk factors for iron deficiency such as prematurity or low birth weight, exclusive breastfeeding without supplementation of iron or introduction of iron-containing foods, or excessive cow’s milk ingestion should be obtained [1]. If an infant has a hemoglobin concentration of less than 11 g/dL or has significant risk factors for iron deficiency, a serum ferritin and CRP (see diagnosis section) should be obtained [1]. If after 1 year of age, a child consumes greater than 24 ounces of cow’s milk or has fewer than two servings per day of iron-containing foods (meats, iron-fortified cereals), we would recommend obtaining a serum hemoglobin for further screening. Frequently milk is not introduced until 1 year of age, and the consumption increases during the 2nd and 3rd year of life. If screening is limited to only the 1-year well-child appointment, children who become iron deficient from excessive cow’s milk intake would be missed. A good diet history can be just as effective as laboratory screening for microcytic anemia. A study evaluating 205 healthy, African-American children living in a low-income setting found that a brief diet history identified children at risk for microcytic anemia 97% of the time [27].

Screening should not just be limited to young children. Several studies have indicated that cognitive impairments are present in adolescents who are iron deficient [11, 12]. Risk factors for iron deficiency in the adolescent population include heavy menses, or other blood loss, such as GI blood loss. Low body weight and malnutrition are also a risk factor for iron deficiency in this population. This may be due to inadequate intake from a vegetarian or vegan diet [21]. Children who are overweight have also been shown to have an increased prevalence of iron deficiency [28]. Additionally, athletes are at risk of iron deficiency and anemia [29]. It is imperative that the general pediatrician screen adolescent patients for these risk factors at the yearly health and wellness evaluation. A CBC and ferritin should then be obtained [28–30].

Clinical Symptoms and Signs of Iron Deficiency Anemia

The classic presentation of iron deficiency anemia in the USA is the presence of microcytic, hypochromic anemia found on screening laboratory during a well-child exam in an asymptomatic young child. Children with more severe anemia may present with pallor, lethargy, poor feeding, irritability, and tachypnea. Children with developmental delay should be screened for iron deficiency. Febrile seizures have been associated with iron deficiency in a number of publications [31]. Exercise intolerance, decreased sports performance, and fatigue can also be symptoms of iron deficiency anemia in the older child. Iron supplementation has been reported to improve athletic performance in patients with iron deficiency anemia [25].

Pica, an intense craving for nonfood items, is a common manifestation of iron deficiency. Examples of pica can include children eating ice, clay, dirt, paper, carpet, starch, and soap. It is possible that a patient may develop concomitant lead poisoning due to iron deficiency-induced pica due to consumption of environmentally contaminated materials such as dried and lead-based paint chips [32]. Pica generally resolves rapidly with replacement of iron. Restless legs syndrome has been associated with iron deficiency in adults and children [33]. Kotagal and Silber demonstrated that serum ferritin levels in children with restless leg syndrome were low in the majority of patients (33% below the 5th percentile and 75% below the median) [34].

Diagnosis and Treatment

Laboratory Testing

Anemia in a pediatric patient is not always attributable to iron deficiency. Many other causes of anemia in childhood exist including anemia secondary to decreased production such as an infiltrative bone marrow process like leukemia or secondary to increased destruction such as a hemolytic anemia. Further studies may be necessary if the patient does not have risk factors or lacks the classic findings of microcytic anemia. Laboratory findings in classic iron deficiency anemia include microcytosis, anemia, elevated red blood cell distribution width (RDW), low red blood cell count, and occasionally thrombocytosis. Patients with microcytic anemia with both a low red blood count (RBC) and low MCV are likely to have iron deficiency, whereas patients with thalassemia will have a normal RBC count and a low MCV. William Mentzer described a Mentzer index: quotient of the MCV divided by the RBC count. If the quotient is less than 13, then iron deficiency anemia is more likely, whereas an index greater than 13 suggests thalassemia [35]. Peripheral smear evaluation demonstrates hypochromia, anisocytosis, and microcytosis. Reticulocyte count will be low if measured but is not required for the diagnosis of iron deficiency anemia .

Classically in general pediatrics in a child with known risk factors and microcytic anemia, iron studies are not necessary. Iron studies may be required for unclear diagnoses. In the event specific testing for iron stores is needed; the three tests that provide the most information regarding iron status include serum ferritin, reticulocyte hemoglobin concentration, and serum transferrin receptor 1 concentration [1]. Serum ferritin is widely used for determination of iron stores, with a value less than 12 μg/L indicating decreased iron stores. However, serum ferritin is an acute phase reactant and increases in the presence of inflammatory states such infection and chronic inflammation or in the presence of malignancy or liver disease. A clinician must utilize a good history and physical exam to assess for the presence of inflammation, and further iron testing is helpful in distinguishing between iron deficiency anemia and anemia of chronic disease where iron utilization is compromised by inflammation. Obtaining a C-reactive protein can be of assistance when interpreting a serum ferritin [1]. Obtaining a serum ferritin for children is a reliable screening test to assess long-term iron stores in the absence of chronic inflammation. The reticulocyte hemoglobin and serum transferrin receptor 1 tests are more accurate than the ferritin, as they are not acute phase reactants, and should be considered in the setting of inflammation [1]. Thus, for a child with microcytic anemia with a chronic disease, we would suggest ordering a serum ferritin and CRP, in addition to a good physical exam, to confirm diagnosis. In a clinically stable child with risk factors for iron deficiency and classic laboratory findings of microcytic anemia with elevated RDW, diagnosis can be confirmed by using a therapeutic challenge by initiating iron supplementation and monitoring response. Reticulocytosis develops within 1 week, and an increase of 1 gm/dL of hemoglobin after 1 month of therapeutic supplementation indicates the presence of iron deficiency anemia. If a therapeutic challenge is performed, the clinician should ensure an adequate dose of iron therapy, that there is no concern for abnormal intestinal iron absorption, and close follow-up [1].

Oral Iron Therapy

Once a diagnosis of iron deficiency anemia is made, iron therapy should be initiated. Oral iron is usually effective, readily available, and inexpensive. The recommended dose is 3–6 mg/kg/day of elemental iron. Ferrous sulfate at 3 mg/kg/day has been shown to yield a greater increase in hemoglobin concentration than iron polysaccharide complex [36]. The milligrams of ferrous sulfate or iron polysaccharide complex should be converted to elemental iron concentration to avoid under dosing. Iron is best absorbed enterally when it is given on an empty stomach with orange juice and not cow’s milk which interferes with iron absorption [37]. Ascorbic acid has been shown to enhance the absorption of nonheme iron [38]. Therefore, it is often recommended that iron be taken with juice or a vitamin C supplement. Additionally, iron should be given as a once-daily dosing. When iron is given in divided doses each day, this has been shown to increase serum hepcidin levels and therefore reduces enteral absorption [39]. Thus far, studies assessing better absorption with alternate day iron dosing have been performed in iron-deficient young women [38]. We have concerns about increasing noncompliance in pediatric patients with alternate day dosing. It is recommended to treat with therapeutic iron for at least 12 weeks [36]. However, we would recommend that to adequately replete iron stores, the 12 weeks should start once a patient’s hemoglobin has normalized. Appropriate response to iron supplementation will show a rise of greater than 1 g/dl of hemoglobin within 2–4 weeks depending upon the degree of anemia. Poor response should be evaluated for compliance or absorption and stools tested for chronic blood loss or malabsorption. An iron absorption test can be performed in clinics to assess the serum iron response to a dose of oral iron. This is a simple test performed by obtaining an iron level when a patient has not eaten anything overnight. A dose of around 4–6 mg/kg of elemental iron is given by mouth. One to 2 hours later, a repeat serum iron level is obtained. If iron has been absorbed, then we expect to see a rise in this level from the previous one. There are very rare mutations that interfere with iron transport, resulting in iron deficiency anemia, such as iron-refractory iron deficiency anemia (IRIDA) . In instances such as these, iron absorptions would be impaired. More commonly absorption is compromised due to underlying disease such as Crohn disease, jejunal feeding, or resection of the duodenum.

Adverse effects of iron therapy are minimal when a dose of 3 mg/kg/day of iron was used, with constipation being the most commonly reported when compared to placebo [40]. Other adverse effects include abdominal pain and diarrhea. We would recommend liberal use of a stool softener such as lactulose or polyethylene glycol over discontinuation or dose modification of the iron supplement if constipation develops. The palatability of oral iron preparations and staining of clothing are sometimes a deterrent to adequate compliance.

In young children found to have iron deficiency anemia, dietary changes are recommended including transitioning from the bottle and stopping or significantly decreasing cow’s milk ingestion. In all children older than 12 months, cow’s milk should be limited to less than 20 oz per day. Lists of iron-rich foods, including meat, Cream of Wheat, prune juice, spinach, raisins, red beans, and iron-fortified cereals, are available. However, increased dietary intake of iron-rich foods alone is inadequate to treat iron deficiency anemia .

Intravenous (IV) Iron Therapy

IV iron is usually considered second-line to oral therapy in the majority of patients. Children with underlying blood loss from gastrointestinal disease or dysfunctional uterine bleeding or inadequate absorption often benefit from IV iron therapy. A number of IV iron forms exist with newer ones offering a safer toxicity profile [26]. Adverse effects may include rash, palpitations, dizziness, myalgias, and chest discomfort. Minor infusion reactions occur in less than 1% of patients and generally resolve by stopping the infusion. More serious anaphylactic reactions are rare. If a patient meets criteria for IV iron, we recommend a referral to pediatric hematology for treatment in a monitored infusion clinic.

Vitamin B12 and Folic Acid Deficiency

Nutritional deficiencies in folic acid and vitamin B12 (also known as cobalamin) can cause impaired DNA synthesis and a form of macrocytic anemia, called megaloblastic anemia. Hematopoietic precursor cells divide rapidly and are therefore susceptible to abnormal DNA synthesis caused by vitamin B12 and folate deficiencies [41].

Both folate and cobalamin are necessary in DNA synthesis, RNA synthesis, and cell duplication [50]. Megaloblastic changes in red blood cell (RBC) precursor cells are noted when DNA is unable to be appropriately synthesized. The cytoplasm in RBC precursors in the bone marrow continues to mature, while nuclear duplication slows, resulting in nuclear-cytoplasmic dyssynchrony [42]. The resulting RBCs are therefore large (macrocytic) and typically oval in shape. In addition to RBC precursors, other hematopoietic cells are affected by B12 and folate deficiencies. Patients can develop thrombocytopenia and neutropenia, with hypersegmented (greater than 5 lobes) neutrophils [43]. The resultant hypercellular bone marrow with dysplastic features can be mistaken for leukemia [42]. While folate and vitamin B12 deficiencies are the most common causes of megaloblastic anemia, certain medications that impair DNA synthesis can also cause megaloblastic anemia [44]. Rarely, megaloblastic anemia can be caused by congenital disorders resulting in folate and vitamin B12 deficiencies [45].

Folate Deficiency

Dietary folate deficiency is becoming increasingly rare in developed countries where folate is supplemented in most common food products like grains and cereals. In the USA, folate deficiency is generally seen in the presence of bowel resection, chronic diarrhea, or chronic hemolytic anemia. The incidence of folate deficiency in an epidemiology study in northern India was 6% in breastfed babies 6–30 months of age and 33% in non-breastfed children of the same age [46].

Pathophysiology

Dietary folates found in plant-based foods and fortified grains are absorbed in the jejunum. Then folate must be reduced to tetrahydrofolate to be biologically active and participate in DNA synthesis. Folate serves as a one-carbon donor and acceptor in many biological pathways, including the production of both purines and pyrimidines, which are fundamental for DNA synthesis. The major role of folate in DNA synthesis is to provide methyl groups which are added to other molecules. A decrease in folate intake can result in folate deficiency in a matter of weeks to months [47].

Etiology

Humans cannot synthesize folate themselves; thus we must obtain folate from sources such as dark green vegetables, fruits, legumes, and animal organs. Boiling or heating these foods can reduce their folate content [48]. Most grains and cereals are now fortified with folate. Individuals with a balanced diet should not become deficient, unless they have increased folate requirements such as in pregnancy, accelerated growth, and chronic hemolysis. Folate deficiency during pregnancy has been associated with neural tube defects in the developing fetus [49]. Those with poor diets, such as in chronic alcoholism or anorexia, and malnourished children are at higher risk for folate deficiency [48]. Folic acid deficiency is seen in infants fed goat’s milk, which has less folate than cow’s milk and supplemented formula [50]. Folic acid deficiency can also occur as a result of poor absorption, as seen in rare congenital disorders such as hereditary folate malabsorption and functional methionine synthesis deficiency or malabsorptive processes like inflammatory bowel disease (IBD), celiac disease, jejunal resection, and chronic diarrhea [51]. Finally, medications such as methotrexate and trimethoprim inhibit the enzymes that convert folate to tetrahydrofolate. Certain anticonvulsants (phenytoin, valproate, and carbamazepine) interfere with folate absorption [52].

Folate requirements are increased in the setting of chronic hemolytic anemias (sickle cell disease, hereditary spherocytosis), hemodialysis, and exfoliative skin disease [52]. These disorders are associated with increased cell turnover and increased DNA synthesis. Folic acid supplementation at a dose of 1 mg daily is helpful to prevent folate deficiency.

Diagnosis

In a patient who presents with macrocytic anemia, serum and erythrocyte levels of folic acid are adequate to diagnose folic acid deficiency; serum levels reflect recent intake, whereas erythrocyte levels are indicative of more chronic folic acid levels 62). Normal serum levels range from 5 to 20 ng/ml, and normal erythrocyte concentration is 150–600 ng/ml [52]. If patients have developed a megaloblastic anemia, RBCs will have an MCV >100 fl, and there are megaloblastic changes on peripheral blood smear and bone marrow. Reticulocyte count is typically low [43].

Treatment

Folate supplementation at 1 mg/day should be started in individuals who are at risk for folic acid deficiency. Folic acid supplementation, either parentally or enterally, should be initiated in patients with folic acid deficiency. Treatment is typically 0.5–1 mg/day for 3–4 weeks [52]. A small 0.1 mg dose for 1 week, with evaluation for hematologic response afterward, can be trialed if the diagnosis is unclear [43]. Once hematologic response is established, maintenance dosing can be continued at the recommended dietary allowance (0.2 mg) [52].

Vitamin B12 Deficiency

Prevalence

The prevalence of vitamin B12 deficiency increases with age with approximately 6% of people over 60 in the USA affected. Individuals with a vegan or vegetarian diet are at increased risk of vitamin B12 deficiency. Breastfed babies of vegan mothers are also at risk [53].

Pathophysiology

Vitamin B12 is released from food by enzymes and acid in the stomach and then complexed with intrinsic factor, which is produced by gastric parietal cells in the stomach [52]. This complex is necessary for absorption in the ileum. Vitamin B12 is then broken down into adenosylcobalamin or methylcobalamin, which are used in DNA and RNA synthesis by serving as cofactors in the conversion of homocysteine to methionine and methyl-malonyl-CoA to succinyl-CoA. Deficiency of B12 can result in folate becoming trapped in the 5-methyl-THF form [41].

Etiology

Like folate deficiency , the most common cause of vitamin B12 deficiency is inadequate dietary intake. The main dietary sources of vitamin B12 include meat, dairy, and eggs; therefore, veganism is a common cause of vitamin B12 deficiency [48]. Deficiency usually takes 1–2 years to develop but can be up to 5 years, due to large stores in the liver [42]. Exclusively breastfed infants of mothers who are vitamin B 12 deficient can develop deficiencies themselves [42]. Signs of deficiency in these babies can appear between 6 and 18 months of life and include macrocytic anemia and loss of motor milestones.

Pernicious anemia is an autoimmune disorder that interferes with the formation of the B12-intrinsic factor complex due to the presence of anti-intrinsic factor or anti-parietal cell antibodies [52]. Pernicious anemia is a common cause of vitamin B12 deficiency when B12 intake is adequate, as it causes decreased absorption of vitamin B12. Pernicious anemia is rare in children. Other gastric pathologies, such as H. pylori infection and bariatric surgeries, decrease the absorption of vitamin B12 by disrupting the release of intrinsic factors [43]. Since B12 is absorbed in the ileum, disorders such as necrotizing enterocolitis which results in bowel resection and subsequent short gut syndrome, celiac disease, IBD, and pancreatic insufficiency can decrease absorption and cause deficiency [42].

Medications including metformin and antacids can also decrease the absorption of vitamin B12 [42]. Finally, rare genetic syndromes that cause decreased vitamin B12 absorption include hereditary intrinsic factor deficiency, Imerslund-Grasbeck (juvenile megaloblastic anemia) syndrome, and inborn errors of cobalamin metabolism [54–56]. Abuse of nitrous oxide can result in severe vitamin B12 deficiency [52].

Signs and Symptoms

All blood elements can show megaloblastic changes; however, erythrocytes show the most marked abnormalities in size and shape, with large oval macrocytes and prominent anisopoikilocytosis. Common laboratory findings also include macrocytic anemia with MCV 100–150 fl and neutrophils with hypersegmentation of their nuclei with greater than 5 lobes [52]. Leucopenia and thrombocytopenia may also be present.

The manifestations of B12 deficiency are not restricted to anemia but often present as neurologic complications [57]. Neuronal effects for vitamin B12 deficiency are partially due to reduced methylation of neuronal lipids and proteins, including myelin basic protein. Myelin basic protein comprises approximately one third of myelin, and demyelination in the setting of vitamin B12 deficiency may explain many of the neurologic manifestations, including paresthesias, numbness, gait abnormalities, cognitive impairment, and neural tube defects in fetuses [58]. The classic neurologic finding in vitamin B12 deficiency is subacute combined degeneration of the dorsal and lateral columns of the spinal cord due to demyelination, associated with ataxia, progressive weakness, and paresthesias which may progress to significant paraplegia [42]. Other non-hematologic manifestations include osteopenia and vascular occlusive disease, due to accumulation of homocysteine [52].

Diagnosis

Serum vitamin B12 levels can be used to evaluate for vitamin B12 deficiency, though there is a high occurrence of both false-positive and