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Do you know which disease fits this month’s case? Then test your knowledge in the quiz below!

What caused the observed thrombocytopenia and production of microcytic red blood cells? Chronic bleeding with iron deficiency
Acute idiopathic thrombocytopenic purpura (ITP) with beta-thalassaemia minor
Myelodysplastic syndrome: refractory anaemia with ring sideroblasts (RARS)
Chronic idiopathic thrombocytopenic purpura (ITP) with iron deficiency

Online version of this month´s case:

The correct answer to February´s Quiz is:

Acute idiopathic thrombocytopenic purpura (ITP) with beta-thalassaemia minor.

Scattergrams and microscopy

Patient history: a 35-year old woman complained about easy bruising and petechia.


Interpretation and Differential Diagnosis

The answer can be inferred from…

  • Absence of anaemia: HGB normal and RBC slightly increased
  • Microcytosis: low MCV and high MicroR%
  • Hypochromia: low MCH and high HYPO-He
  • Slight erythrocytosis: high RBC count
  • No anisocytosis: normal RDW-SD
  • Chronic ineffective erythropoiesis: low RET-He and normal Delta-He
  • Severe thrombocytopenia: extremely low PLT-I and PLT-F
  • Effective megakaryopoiesis: increased IPF


Case history

A 35-year old woman visited her physician with bruises and petechia. The complete blood count results showed a severe thrombocytopenia and RBC abnormalities.

Case results

A severe, potentially life-threatening thrombocytopenia was detected. In combination with a high immature platelet fraction (IPF) this indicated an increased destruction of platelets in the peripheral blood. This could indicate a microangiopathic haemolytic anaemia such as thrombotic thrombocytopenic purpura or haemolytic uraemic syndrome, or an autoimmune thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) or drug-induced immune thrombocytopenia. Microangiopathic haemolytic anaemia could be excluded because RBC fragments were not observed (FRC# and FRC% were only slightly increased, the ‘Fragments?’ flag was absent and fragments were not detected in the peripheral blood smear). Nevertheless, abnormal combinations of RBC indices were notable:

1) Extreme microcytic hypochromic erythropoiesis without anisocytosis (RDW-SD < 42 fL), combined with a normal HGB,

2) Slightly increased erythrocyte and reticulocyte counts, a low haemoglobinisation of reticulocytes (RET-He) and a normal Delta-He.

These two combinations of abnormal RBC indices indicate chronic (long-term) ineffective erythropoiesis. Acute microcytic hypochromic anaemia such as iron deficiency anaemia would have been associated with anisocytosis and a negative Delta-He (1). It could also be excluded here based on several different formulas, for example:

1) The Green and King index (MCV2 * RDW-CV / HGB) shows a value of 53 here – values below 73 point to β-thalassaemia minor (2),

2) The Urrechaga index (MicroR - HYPO-He - RDW-CV) shows a value of 20.1 here – values above -5.1 also point to β-thalassaemia minor (3).

In addition, reticulocyte parameters such as RET-He and Delta-He can distinguish chronic from acute causes: the observed low RET-He combined with the normal Delta-He value indicated a chronic erythropoietic inefficiency as seen in diseases such as β-thalassaemia or myelodysplastic syndromes, while a low RET-He combined with a low Delta-He would have indicated a recent deterioration in erythropoiesis. The peripheral blood smear review that was consequently performed revealed a poikilocytosis with the presence of target cells and stomatocytes. Furthermore, erythroid hyperplasia was observed in the bone marrow smear indicating increased erythropoiesis (also indicated by the slightly increased RET#). The diagnosis of β-thalassaemia in this patient was confirmed by haemoglobin analysis with electrophoresis, which revealed β chain insufficiency.


The following answers are incorrect for the described reasons

Chronic bleeding with iron deficiency

A chronic bleeding can have many causes such as nose bleedings, haemorrhoids, ulcers and tumours of the intestine, kidney or bladder. It may go undetected for a long time when blood loss is slow and not obvious (for example, lower gastrointestinal bleedings). A chronic bleeding results in iron deficiency when the intake of iron is not enough to compensate for iron loss caused by the bleeding. RET-He would be decreased as observed here. However, the body also responds to a chronic bleeding by increasing erythropoiesis, which leads to reticulocytosis, and by directing extra fluid into the vascular space to keep the blood volume constant, which leads to decreased HGB and RBC values. Neither a reticulocytosis nor decreased HGB or RBC was observed here. In addition, the Green and King index (MCV2 * RDW-CV / HGB = 53) is below 73 and the Urrechaga index (MicroR - HYPO-He - RDW-CV = 20.1) is above -5.1, pointing to β-thalassaemia rather than an iron deficiency. For these reasons this diagnosis cannot be correct.


Myelodysplastic syndrome: refractory anaemia with ring sideroblasts (RARS)

The myelodysplastic syndromes comprise a heterogeneous group of malignant haematopoietic stem cell disorders characterized by dysplastic and ineffective blood cell production. Refractory anaemia with ring sideroblasts (RARS) is characterized by anaemia, erythroid dysplasia and ring sideroblasts. Anaemia is usually macrocytic or normocytic, and the number of reticulocytes is low. Patients may also have thrombocytopenia or neutropenia. Although a thrombocytopenia was observed here, RARS can be excluded because none of the other characteristics was found. This patient had increased reticulocyte counts and microcytic RBC, and was not anaemic. In addition, RET-He was low here while erythropoiesis is decreased in RARS leading to high iron levels, which in some cases may progress into iron overload. RARS could therefore be excluded.


Chronic idiopathic thrombocytopenic purpura (ITP) with iron deficiency

When ITP persists for more than twelve months, it is called chronic ITP. Spontaneous remission only occurs in 10-20% of patients. While platelet destruction is increased in acute ITP, chronic ITP is associated with reduced platelet production, possibly because the activated immune system damages megakaryocytes, which have similar surface proteins as platelets. However, the IPF was increased here indicating that megakaryopoiesis was not affected. In addition, the Green and King index (MCV2 * RDW-CV / HGB = 53) is below 73 and the Urrechaga index (MicroR - HYPO-He - RDW-CV = 20.1) is above -5.1, pointing to β-thalassaemia rather than iron deficiency so this diagnosis could be excluded as well.

Underlying Disease

Thrombocytopenia caused by antibodies

Antibodies reacting with cell surface antigens on thrombocytes may result in accelerated platelet destruction and subsequent thrombocytopenia. Antibody-induced thrombocytopenia can result from:

1. The production of alloantibodies after transfusion or pregnancy:

a.     Neonatal alloimmune thrombocytopenia
b.     Post-transfusion purpura


2.     The production of autoantibodies (autoimmune thrombocytopenia: glycoprotein-specific autoantibodies binding to autologous platelets):

a.     Acute ITP usually affects children after a viral infection but is also occasionally observed after vaccination. Petechia or extensive bleeding are the main clinical manifestations and acute ITP is fully reversible.

b.     Chronic ITP mostly persists for more than twelve months and spontaneous remission only occurs in 10-20% of patients. There are two forms of chronic autoimmune thrombocytopenic purpura: idiopathic without concurrent primary disease or a secondary form resulting from haematological diseases such as non-Hodgkin lymphoma.

c.      In drug-induced immune thrombocytopenia, drugs cause the production of antibodies that bind to epitopes on platelets, which leads to their destruction. This typically only happens during the presence of the drug.

d.     Heparin-induced thrombocytopenia type II is caused by antibodies that recognize an epitope on platelet factor 4 bound to heparin, 7-10 days after heparin therapy (or immediately after re-exposure). When complexes of a certain size are generated, platelets clots are formed and platelet counts may drop below 20,000/µL. Bleeding complications are rare while arterial thromboembolic complications are common.


Idiopathic thrombocytopenic purpura (ITP)

ITP is an autoimmune haematological disorder in which autoantibodies against platelet antigens induce accelerated platelet destruction, leading to a reduction in peripheral blood platelets. ITP causes a characteristic purpuric rash and a tendency to bleed, for example from the nose or periodontal gums. It is difficult to distinguish ITP from other causes of thrombocytopenia so its diagnosis is a process of exclusion. Megakaryopoietic activity of the bone marrow is typically enhanced in acute ITP.



Thalassaemia is an autosomal heritable haemolytic anaemia caused by defective globin chain synthesis. β-Thalassaemia is a quantitative disorder of the synthesis of β-globin chains: over 200 mutations leading to a reduced or fully diminished production of β chains have been identified and the type of mutation can influence the severity of the disease. The lack of β chains is compensated by the insertion of additional α chains. This creates unstable α chain tetra­mers that, depending on their concentration, cause the premature destruction of erythroblasts and subsequent ineffective erythropoiesis. Contrary to α-thalassaemia, β-thalassaemia symptoms don’t manifest in the first few months of life, because β chains are not synthesised until after birth.

The clinical expression of thalassaemia depends on the extent of the genetic defect and whether it’s homozygous or heterozygous. The severity of the clinical symptoms is used to classify thalassaemias into major, intermedia and minor. β-Thalassaemia major is a life-threatening condition associated with severe anaemia. Lifelong blood transfusions are required and if untreated, β-thalassaemia major leads to death in the first decade. β-Thalassaemia major arises when both alleles of the β-globin gene are affected by mutations leading to the production of fewer or no β chains. β-Thalassaemia minor, or trait, is usually asymptomatic, but in some cases it can lead to mild anaemia. It’s a heterozygous condition, where one allele of the β-globin gene functions normally while mutations in the second allele result in the production of fewer or no β chains. β-Thalassaemia intermedia patients present with symptoms that vary from mild to severe. Most patients have moderate anaemia that does not require regular transfusions. Patients present later than those with β-thalassaemia major. Like β-thalassaemia minor, β-thalassaemia intermedia is usually heterozygous and classifying the disease as intermedia depends on the severity of clinical manifestations.


β-Thalassaemia minor (trait)

Patients with β-thalassaemia minor can be asymptomatic or present with anaemia-related symptoms such as tiredness, weakness, numbness in the extremities, a weak immune system and depression. In some cases, enlargement of the spleen may also be noted. β-Thalassaemia minor resembles iron deficiency anaemia and differentiation between the two conditions is essential for a correct diagnosis and therapy. Long term inadequate treatment of thalassaemia with iron supplement may cause problems, including organ damage.

The first CBC results can already be useful for differentiating thalassaemia minor and iron deficiency. For example, in iron deficiency anaemia, RBC is usually normal to slightly increased, while it is elevated in mild thalassaemia. Another informative parameter is the red cell distribution width (RDW). As iron deficiency anaemia progresses, each successive wave of new RBC gets smaller and smaller, which results in a high RDW. In contrast, in thalassaemia all RBC are uniformly small, and the RDW is low. MircoR and HYPO-He are other useful parameters to differentiate between thalassaemia and iron deficiency anaemia: in iron deficiency, the number of microcytes and the fraction of hypochromic RBC (HYPO-He) are equally increased resulting in a MicroR to HYPO-He ratio of 1.0 or slightly below 1.0, whereas in thalassaemia the ratio MicroR to HYPO-He is mostly increased (4). Several different formulas have been proposed that take into account RBC parameters from the blood count:

Green and King index (MCV2 * RDW-CV / HGB)

< 73 → β-thalassaemia minor

> 73 → iron deficiency

Mentzer index (MCV / RBC)

< 13 → β-thalassaemia minor

> 13 → iron deficiency

Urrechaga index (MicroR - HYPO-He - RDW-CV)

> -5.1 → β-thalassaemia minor
< -5.1 → iron deficiency

Recently it has been demonstrated by Urrechaga et al. that the index using the Sysmex parameters MicroR, HYPO-He, RDW-CV shows a very good sensitivity and specificity (3). In this case, the values of the Green and King index (=53), Mentzer index (=12.8) and Urrechaga index (=20.1) all indicate thalassaemia and not iron deficiency. Normal haemoglobin levels, the absence of NRBCs and a very mild manifestation in a young adult all suggest a thalassaemia minor subtype.



  1. Urrechaga E et al. (2013): Erythrocyte and reticulocyte indices in the assessment of erythropoiesis activity and iron availability. Int J Lab Hematol. 35(2):144-9
  2. Green R and King R (1989): A new red cell discriminant incorporating volume dispersion for differentiating iron deficiency anemia from thalassemia minor. Blood Cells, 15:481-495
  3. Urrechaga E et al. (2011): The role of automated measurement of RBC subpopulations in differential diagnosis of microcytic anemia and β-thalassemia screening. Am J Clin Pathol. 135:374-379
  4. Urrechaga E et al. (2011): Erythrocyte parameters in iron deficiency and thalassemia. J Clin Lab Anal. 25:223-228


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