Dr. Bharat Agarwal *
Consultant Pediatric Hematologist-Oncologist Head, Dept., of Pediatric Hematology-Oncology, B.J.Wadia Hospital for Children, Parel, Mumbai - 400 012 (INDIA) *
When a patient presents with anemia, it is important to establish whether the problem is due to a single line (e.g., red blood cells) or a multiple cell line (i.e., red cell, white cell, and platelets). A two or three cell line problem usually indicates bone marrow involvement (e.g., aplastic anemia, leukemia), immunologic disorder (e.g., connective tissue disease, acquired immunodeficiency syndrome [AIDS]), peripheral destruction of cells (e.g., immunoneutropenia, idiopathic thrombocytopenic purpura [ITP], or immune hemolytic anemia singly or in combination), or sequestration of cells (e.g., hypersplenism). Table 1-1 presents an etiologic classification of anemia and the diagnostic features in each case.

Table 1-1 Etiologic Classification and major Diagnostic Features of Anemia in Children.

Etiologic classification Diagnostic features
1.Impaired red cell formation
  A. Deficiency
  • Decreased dietary intake (eg excessive milk-iron deficiency anemia,
    vegan-vitamin B12 deficiency
  • Increased demand, e.g., growth (Iron),
  • Hemolysis (folic acid)
  • Decreased absorption Specific : intrinsic
    factor lack (Vitamin B12) Generalized:
    malabsorption syndrome
    (e.g., folic acid, iron)
  • Increased loss

Acute : hemorrhage (iron)
Chronic : gut bleeding (iron)
1. Iron deficiency
Hypochromic, microcytic red cells; low MCV, low MCH, low MCHC high RDWa, a low serum ferritin, high FEP, guaiac positivity
2. Folate deficiency
Macrocytic red cells, high MCV, high RDW, megaloblastic marrow, low Serum and red cell folate
3.Vitamin B12 deficiency
Macrocytic red cells, high MCV, high RDW, megaloblastic marrow, low Serum B12, gastric acidity, Schilling test
4. Vitamin C deficiency Clinical - Scurvy
5. Protein deficiency Kwashiorkor
6. Vitamin B6 deficiency Hypochromic red cells, sideroblastic bone marrow, high serum ferritin
7. Thyroxine deficiency Clinical cretinism, low T4, high TSH

B. Bone marrow failure
1. Failure of a single cell line Limb abnormalities, absent megakaryocytes
a. Megakaryocytes
1. Amegakaryocytic thrombocytopenic purpura
Limb abnormalities, absent megakaryocytes
b. Red cell precursors
1. Congenital red cell aplasia (Diamond- Blackfan anemia)
2. Acquired red cell aplasia (transient erythroblastopenia of childhood [TEC])
Absent red cell precursors

Absent red cell precursors - temporary
c. White cell precursors
3. Congenital neutropenia
Neutropenia, recurrent infection

3. Failure of all cell lines (produces aplastic anemia characterized by pancytopenia and acellular
or hypocellular marrow)
a. Congenital
(1) Fanconi's anemia
(2) Familial without anomalies
(3) Dyskeratosis congenita
Multiple congenital anomalies. Chromosomal breakage
Familial history, no congenital anomalies
Marked mucosal and cutaneous lesions
b. Acquired
(1) Idiopathic
(2) Secondary
(3) Infiltration
a. De novo (e.g., leukemia)

b.Secondary(e.g. neuroblastoma,lymphoma
No identifiable cause
History of exposure to drugs, radiation, household toxins, infections, associated immunologic diseases
Bone marrow: morphology cytochemistry, immunologic markers, cytogenetics.
VMA, skeletal survey, bone marrow
c. Dyshematopoietic anemia
(decreased erythropoiesis, decreased iron utilization)
1. Infection Evidence of systemic illness
2. Renal failure and hepatic disease BUN and liver function tests
3. Disseminated malignancy Clinical evidence
4. Connective tissue disease Rheumatoid arthritis.

II. Blood loss

III. Hemolytic anemia  
A. Corpuscular  
1. Membrane defects (spherocytosis, elliptocytosis) Morphology, osmotic fragility
2. Enzymatic defects (pyruvate kinase, G6PD) (spherocytosis, elliptocytosis) Autohemolysis, enzyme assays
3. Hemoglobin defects
a. Heme
b. Globin
(1) Qualitative (e.g., sickle cell)
(2) Quantitative (e.g. thalassemia)
Hb electrophoresis
HbF, A2 content
B. Extracorpuscular
1) Immune
a. Isoimmune
b. Autoimmune

Secondary Immunologic disorder (e.g. lupus)
  • One cell line (e.g., red cells)
  • Multiple cell line (e.g., white blood cells, platelets)
Coombs' test

Coomb's test, antibody identification
Decreased C3, C4, CH50, positive ANA

Anemia: Coombs, positive
Neutropenia-immunoneutropenia, thrombocytopenia-ITP
2) Non-immune (idiopathic, secondary)


The blood smear is very helpful in the diagnosis of anemia. It establishes whether the anemia is hypochromic, normocytic or macrocytic or shows specific morphologic abnormalities (e.g., spherocytes, sickle cell, or target cells). The mean corpuscular volume (MCV) confirms the findings on the smear with reference to the red cell size, for example microcytic (< 7 µm), macrocytic (> 8.5 µm) or normocytic (7.2-7.9 µm). Table 1-2 lists the differential diagnostic considerations based on specific red cell morphologic abnormalities.

Table 1-2. Specific Red Cell Morphologic Abnormalities

  1. Target Cells

    Increased surface/volume ratio· Thalassemia

    • Hemoglobinopathies· Hb AC or CC· Hb SS, SC, S-Thal

    • Liver disease· Postsplenectomy or hyposplenic states

    • Severe iron deficiency· Hb E (heterozygote and homozygote)

    • LCATdeficiency : congenital disorder of lecithin/cholesterol acyltransferase deficiency (corneal opacifications, proteinuria, target cells, moderately severe anemia)

    • Abetalipoproteinemia.

  2. Spherocytes

    Decreased surface/volume ratio, hyperdense (>MCHC)

    • Hereditary spherocytosis

    • ABO incompatibility: antibody-coated fragment of RBC membrane removed

    • Autoimmune hemolytic anemia : antibody-coated fragment of RBC membrane removed

    • Microangiopathic hemolytic anemia (MAHA) : fragment of RBC lost after impact with abnormal surface

    • SS disease : fragment of RBC removed in reticuloendothelial system

    • Hypersplenism

    • Burns : fragment of damaged RBC removed by spleen

    • Post transfusion

    • Pyruvate kinase deficiency

    • Water-dilution hemolysis : fragment of damaged RBC removed by spleen

  3. Acanthocytes (spur cells): Cells with 5-10 spicules of varying length; spicules irregular in space and thickness, with wide bases; appear smaller than normal cells because they assume a spheroid shape.

    • Liver disease· Disseminated intravascular coagulation (and other MAHA)

    • Postsplenectomy or hyposplenic state

    • Vitamin E deficiency

    • Hypothyroidism

    • Abetalipoproteinemia : rare congenital disorder; 50-100% of cell acanthocytes; Associated abnormalities (fat malabsorption, retinitis pigmentosa, neurologic abnormalities)

    • Malabsorptive states

  4. Echinocytes (burr cells): 10-30 spicules equal in size and evenly distributed over RBC surface; caused by alteration in extracellular environment.

    • Artifact

    • Uremia

    • Dehydration

    • Liver disease

    • Pyruvate kinase deficiency

    • Peptic ulcer disease or gastric carcinoma

    • Immediately after red cell transfusion

    • Rare congenital anemias due to decreased intracellular potassium

  5. Pyknocytes

    Distorted, hyperchromic, contracted RBC, can be similar to echinocytes and acanthocytes.

  6. Schistocytes: Helmet, triangular shapes, or small fragments. Caused by fragmentation upon impact with abnormal vascular surface (e.g., fibrin stand, vasculitis, artificial surface in circulation)

    • Disseminated intravascular coagulation (DIC)

    • Severe hemolytic anemia (e.g., G6PD deficiency)

    • Microangiopathic hemolytic anemia· Hemolytic uremic syndrome

    • Prosthetic cardiac valve, abnormal cardiac valve, cardiac patch, coarctation of the aorta

    • Connective tissue disorder (e.g., systemic lumps erythematosus [SLE])

    • Kasabach-Merritt syndrome

    • Purpura fulminans

    • Burns (spheroschistocytes as a result of heat)

    • Thrombotic thrombocytopenic purpura

    • Hemograft rejection

    • Uremia, acute tubular necrosis, glomerulonephritis

    • Malignant hypertension· Systemic amyloidosis

    • Liver cirrhosis

    • Disseminated carcinomatosis

    • Chronic relapsing schistocytic hemolytic anemia.

  7. Elliptocytes: Elliptical cells, normochromic; seen normally in less than 1% of RBC; larger numbers occasionally seen in a normal patient· Hereditary elliptocytosis

    • Iron deficiency (increased with severity, hypochromic)

    • SS disease· Thalassemia major· Severe bacterial infection

    • SA trait

    • Leukoerythroblastic reaction

    • Megaloblastic anemias

    • Any anemia may occasionally present with up to 10% elliptocytes

    • Malaria

  8. Teardrop cells: Shape of drop, usually microcytic, often also hypochromic

    • Newborn

    • Thalassemia major

    • Leukoerythroblastic reaction

    • Myeloproliferative syndromes

  9. Stomatocytes:

    Has a slit like area of central pallor· Normal (in small numbers)

    • Hereditary stomatocytosis

    • Artifact

    • Thalassemia

    • Acute Alcoholism

    • Rh null disease (absence of Rh complex)

    • Liver disease· Malignancies

  10. Nucleated red blood cells

    Not normal in the peripheral blood beyond the first week of life·

    • Newborn (first 3-4 days)

    • Intense bone marrow stimulation

    • Hypoxia (especially postcardiac arrest)

    • Acute bleeding§ Severe hemolytic anemia (e.g., thalassemia, SS hemoglobinopathy)

    • Congenital infections (e.g., sepsis, congenital syphilis, cytomegalovirus, rubella)

    • Postsplenectomy or hyposplenic states : spleen normally removes nucleated RBCs

    • Leukoerythroblastic reaction, seen with extramedullary hematopoiesis and bone marrow replacement; most commonly leukemia or solid tumor

    • Fungal and Mycobacterial infection may also do this; associated teardrop red cells,

    • 10,000-20,000 white blood cells (WBCs) with small to moderate numbers of metacytes, myelocytes, and promyelocytes;

    • Thrombocytosis with large bizarre myelo platelets

    • Megaloblastic anemia

    • Dyserythropoietic anemias.

  11. Blister cells: Red cell area under membrane, free of hemoglobin, appearing like a blister

    • G6PD deficiency (during hemolytic episode)

    • SS disease

    • Pulmonary emboli

  12. Basophilic stippling: Coarse or fine punctuate basophilic inclusions that represent aggregates of ribosomal RNA

    • Hemolytic Anemias (e.g., thalassemia trait)

    • Iron-deficiency anemia

    • Lead poisoning

  13. Howell-Jolly bodies: Small, well-defined, round, densely stained inclusions; 1 µm in diameter, eccentric in location

    • Postsplenectomy or hyposplenia

    • Newborn

    • Megaloblastic anemias

    • Dyserythropoietic anemias

    • A variety of types of anemias (rarely iron-deficiency anemia, hereditary spherocytosis)

The MCV and reticulocyte count are helpful in the differential diagnosis of anemia. An elevated reticulocyte count suggests chronic blood loss or hemolysis; a normal or depressed count suggests impaired red cell formation.

The reticulocyte count must be adjusted for the level of anemia to obtain the reticulocyte index, a more accurate reflection of erythropoiesis. In patients with bleeding or hemolysis, the reticulocyte index should be at least 3%, whereas in patients with anemia due to decreased production of red cells, the reticulocyte index is less than 3% and the frequency is less than 1.5%.

Reticulocyte index = Reticulocyte X Patient's hematocrit/Normal hematocrit

For example : Reticulocyte count 6%, hematocrit, 15, then

Reticulocyte index =(6 X 15)/45 = 2%

The MCV and red cell distribution width (RDW) indices, available from automated electronic blood-counting equipment, are extremely helpful in defining the morphology and the nature of the anemia and have led to a new classification based on these indices.

In more refractory cases of anemia, bone marrow examination may be indicated. A bone morrow smear should be stained for iron to estimate iron stores and to diagnose iron deficiency anemia.

The investigation of anemia entails the following steps:

  1. Detailed history and physical examination (see Table 1-1).
  2. Complete blood count, to establish whether the anemia is the result of a one cell line (red cell line only) or a three cell line problem (abnormality of red cell count, white blood cell count, and platelet count).
  3. Determination of the morphologic characteristics of the anemia based on blood smear (Table 1-2) and consideration of the MCV and RDW and morphologic consideration of white blood cell and platelet morphology.
  4. Bone morrow aspiration, if required, to examine erythroid, myeloid, and megakaryocytic morphology to determine whether there is normoblastic, megaloblastic, or sideroblastic erythropoiesis and to exclude marrow pathology (e.g., aplastic anemia, leukemia, and benign or malignant infiltration of the bone marrow.
  5. Determination of underlying cause of anemia by additional tests.
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