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May 2014 - Test your knowledge in the quiz!

Do you know why this 24-year old man is constantly tired? Iron deficiency anaemia
Crohn’s disease
Beta-thalassaemia intermedia
Toxoplasmosis and thalassaemia minor

Online version of this month`s case:

THE CORRECT ANSWER TO MAY’S QUIZ IS:

Thalassaemia minor and toxoplasmosis

Scattergrams and microscopy

„Patient history: a 24-year old patient came to the hospital complaining about general tiredness.”

 

WDF scattergram
WNR scattergram
RET scattergram
RBC histogram
Peripheral blood smear
Peripheral blood smear

Table

INTERPRETATION AND DIFFERENTIAL DIAGNOSIS

The answer can be inferred from…

 

Case history

A young male complaining of fatigue and malaise came to a general practitioner. His cervical lymph nodes were swollen and sensitive during palpation, but the patient had no fever. Haematological malignancy had to be urgently ruled out, as it is a common cause of sensitive lymph nodes and fatigue. A blood sample was taken for analysis.

Case results

The differential WBC counts suggested an inflammatory reaction, whereas the elevated differential counts for monocytes and lymphocytes could indicate an infectious or non-infectious inflammation reaction or a malignant condition. However, the “Blasts/Abn Lympho?” flag was not triggered and the absence of blasts was later confirmed by a smear review, which also revealed the absence of morphological anomalies of WBCs. Eosinophilia may be present in cases of allergy, parasitic infection and non-infectious inflammation response, and the elevated absolute eosinophil count in this case indicates that a bacterial or viral infection is unlikely in this patient.

Besides the leukocytosis, the abnormal combinations of RBC indices were notable: 1) extreme microcytic hypochromic erythropoiesis without anisocytosis (RDW-SD < 42 fL), but with a slightly decreased to normal HGB, 2) the combination of extreme high erythrocyte counts without reticulocytosis, a low RET-He but a normal Delta-He. These two combinations of abnormal RBC indices indicate chronic (long term) ineffective erythropoiesis. Beside a low MCV, other microcytic hypochromic anaemias like absolute iron deficiency anaemia would show a lower HGB, anisocytosis and a negative Delta-He (1). Microcytic hypochromic conditions can be differentiated based on several different formulas that take into account red blood cell parameters from the blood count, for example:

1) The Green and King index (MCV2 * RDW-CV / HGB) shows a decreased value of 57 here (values below 73 point to β-thalassaemia minor) (2),

2) The Urrechaga index (MicroR - HYPO-He - RDW-CV) shows an increased value of 14 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: low haemoglobinisation of reticulocytes depicted by low RET-He and normal Delta-He values are indicative of chronic erythropoietic inefficiency as observed in diseases such as β-thalassaemia intermedia/major or MDS. On the other hand, a low RET-He combined with a low Delta-He indicates a recent deterioration of erythropoiesis.

The observed leukocytosis and the high RBC count are not typical for the MDS. In addition, leukocytosis and a microcytic anaemia together suggest the anaemia of chronic disease (ACD), which is a functional iron deficiency due to inflammation. However, by taking into account multiple parameters and using the discriminant formulas (2-3), both ACD and IDA could be ruled out, suggesting that the patient has β-thalassaemia (for details see the section “Underlying disease”). Smear review confirmed the microcytic hypochromic anaemia in this patient and revealed significant numbers of red blood cells with basophilic stippling, which is present in cases of ineffective erythropoiesis and is characteristic for thalassaemia and rare cases of iron deficiency (4). The diagnosis of β-thalassaemia in this patient was confirmed by haemoglobin analysis by electrophoresis, which revealed β-chain insufficiency.

Additional test had to be carried out to find the cause of leukocytosis. In this case, the patient suffered from a primary Toxoplasma infection, which is known to cause lymphocytosis, monocytosis and, in some cases, eosinophilia, all due to inflammation triggered by the parasites (for details see the section “Underlying disease”).

 

The following answers are incorrect for the described reasons

Iron deficiency anaemia (IDA)

Anaemia is a common condition, particularly in young women and in the geriatric population, and it is a significant public health problem in developing countries. Anaemia is defined by the World Health Organisation as a haemoglobin concentration below 12 g/dL in women and below 13 g/dL in men. Iron deficiency is the most frequent cause of anaemia (29% of all causes) and remains the most common cause of microcytosis, followed by thalassaemia. Iron deficiency states are characterized by hypochromic, microcytic anaemia. Patients suffer from fatigue, paleness, tachycardia and shortness of breath.

Iron metabolism is finely tuned to regulate intestinal absorption and serum iron levels. Iron deficiency is defined as a low serum iron concentration, elevated transferrin iron binding capacity (as a response to deficiency) and a low serum ferritin concentration (reflecting low iron stores). Iron deficiency anaemia (IDA) requires a differential diagnosis from anaemia of chronic disease (ACD), β-thalassaemia and myelodysplastic syndromes (MDS), which can all present with microcytic and hypochromic red blood cells (RBC).
However, IDA alone could not explain the lymphocytosis, monocytosis and eosinophilia in this patient. In addition, a combination of RBC indices from the blood count, as found in discriminant scores such as the Green and King index (2) or the Urrechaga index (3), can differentiate between IDA and β-thalassaemia. In this case, iron deficiency anaemia could be excluded based on both indices (for details see the part “Underlying disease”).

 

Crohn’s disease (5)

Crohn’s disease (CD) belongs to a group of conditions known as inflammatory bowel diseases. It is an autoimmune disorder with a genetic predisposition and the first manifestation usually at the age between 15 and 30 years. CD affects the gastrointestinal tract, most often the ileum and upper part of the colon. The symptoms include abdominal pain, fever, fatigue, persistent diarrhoea or constipation, weight loss, mouth ulcers and joint pain.

Approximately one third of CD cases present with anaemia. It is widely accepted that the causes of anaemia in CD are rather mixed, with a portion resulting from iron deficiency (IDA: iron stores are empty) and a portion resulting from anaemia of chronic disease, which causes functional iron deficiency or FID: iron stores are full, but the iron is not available for erythropoiesis. Some patients are affected by IDA and FID simultaneously. Anaemia of chronic disease develops due to the action of inflammatory cytokines, which induce the production of hepcidin in the liver. Hepcidin is known to inhibit duodenal absorption of iron as well as iron release from macrophages. Ferroportin, which is important for exporting iron from enterocytes, hepatocytes and macrophages, is also downregulated by proinflammatory cytokines, further reducing the release of iron from macrophages (6). Both IDA and FID manifest as hypochromic microcytic anaemia. In rare cases of CD, malabsorption of cobalamin and/or folic acid in the ileum leads to the vitamin B12 and/or folate deficiency anaemia, which is characterised by macrocytosis. In this case, Crohn’s disease with vitamin B12 or folate deficiency can therefore be excluded.
Differentiation between IDA and FID in CD can be made using an algorithm developed by Thomas et al. (‘Thomas-Plot’) (7). The measurements required are RET-He, the soluble transferrin to log ferritin ratio (sTFR / log ferritin: sTFR-F index) and the C-reactive protein concentration. In addition, microcytosis is not as prominent in FID as in IDA or thalassaemia. IDA and anaemia of chronic disease, associated with Crohn’s disease, can be excluded in this case based on the Green and King or Urrechaga index (see below).

Chronic inflammation of the bowel could explain leukocytosis observed in this patient, but the combination of the red blood cell indices does not correspond to the anaemia of chronic disease, iron deficiency anaemia or vitamin B12 anaemia, associated with this condition.

 

β-Thalassaemia intermedia

Thalassaemia intermedia is a term used to define a group of patients with β-thalassaemia in whom the clinical severity of the disease is somewhere between the mild symptoms of the β-thalassaemia minor (see below) and the severe manifestations of β-thalassaemia major. The diagnosis is a clinical one and is based on the patient’s ability to maintain a satisfactory haemoglobin level of 6-8 g/dL at the time of diagnosis without the need for regular blood transfusions. Patients present with symptoms later than those with β-thalassaemia major, but usually the condition worsens over time and the patients require blood transfusions later in life.
Some common problems in this condition are an enlarged spleen, inadequate bone development, weak immune and endocrine systems, and excess absorption of iron in the gut, which may lead to iron overload and organ damage. Patients often undergo splenectomy to eliminate the filtering out of red blood cells by the spleen.
Apart from the clinical manifestations and HGB levels, the presence of nucleated red blood cells (NRBC) helps to differentiate between β-thalassaemia intermedia and minor in many cases. Especially in splenectomised patients, the levels of NRBCs are high (8).
In this case β-thalassaemia intermedia can be excluded because of nearly normal haemoglobin levels, late manifestation of the microcytic anaemia and, to some extent, the absence of NRBCs. Apart from that, β-thalassaemia intermedia alone could not explain lymphocytosis, monocytosis and eosinophilia in this patient.

 

 

UNDERLYING DISEASE

β-Thalassaemia minor (trait)

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 lacking β-chains are compensated for 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 thereby an ineffective erythropoiesis. Contrary to α-thalassaemia, β-thalassaemia does not 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 transfusion is 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 chain 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-chain gene functions normally, while mutations in the second allele results 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.  β-Thalassaemia intermedia is usually heterozygous, and classifying a disease as “intermedia” depends on the severity of clinical manifestations.

β-Thalassaemia minor can either be generally asymptomatic or present with anaemia-related symptoms such as tiredness, weakness, numbness in the extremities, weak immune system and depression. In some cases, enlargement of the spleen may also be noted. β-Thalassaemia minor resembles iron deficiency anaemia (IDA) 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.

Already the first CBC results can be useful for differentiating thalassaemia minor and iron deficiency. For example, in IDA, the 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 IDA progresses, each successive wave of new red blood cells gets smaller and smaller, which results in a high RDW. In contrast, in thalassaemia all red blood cells are uniformly small, and the RDW is low. MircoR and HYPO-He are other useful parameters to differentiate between thalassaemia and IDA: in IDA, 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 (9). Several different formulas have been proposed that take into account red blood cell 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 (=57), Mentzer index (=9.7) and Urrechaga index (=14) 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.

 

Toxoplasmosis (10)

Toxoplasmosis is a parasitic infection caused by a protozoan known as Toxoplasma gondii. Toxoplasma has a complex life cycle which involves three different infective stages of the parasite and replication in intermediate and definitive hosts. The infective stages are crescent-shaped cells, approximately 5 µm long.

Felines, including wild animals and domestic cats, are the definitive host of the parasite. When a cat consumes an infected animal, which is an intermediate host of Toxoplasma – a mouse or a bird, for example – the parasites enter the intestinal cells in the gut of the cat, where they undergo sexual replication and are shed in large numbers with the faeces. In the external environment these parasites form spores, which are very robust and can survive for a long time. Intermediate hosts, which include virtually all warm-blooded animals, can be infected by ingesting the spores. In the intermediate host parasites exit the spores, replicate asexually in the intestinal cells and then disseminate in the body with the blood flow. Disseminated parasites form cysts in body tissues, predominantly muscle and brain, and become a source of infection for carnivores.

Humans most often become infected by consuming undercooked meat or unwashed vegetables. Coming into contact with the faeces of infected domestic cats or gardening also poses a risk of infection. Water is a major source of contamination in developing countries. In rare cases, Toxoplasma may be transmitted with a transplanted organ from an infected donor.

It is generally assumed that approximately 25 to 30% of the world’s human population is infected by Toxoplasma. Eighty percent of cases of primary infection with Toxoplasma are asymptomatic. In the remaining cases, patients may experience fever or cervical lymphadenopathy, sometimes associated with fatigue and asthenia. Lymphadenopathy and asthenia may persist for several weeks, resembling infectious mononucleosis or Hodgkin’s lymphoma. One third of the patients presents with eosinophilia. During the primary infection, Toxoplasma causes a local inflammation in the gut, which attracts neutrophils and other phagocytic cells, leading to an immunological response to the infection. However, at the same time the parasites are capable to supress the immune response by interfering with interleukin production by macrophages and to prevent apoptosis of phagocytes with ingested parasite cells. This mechanism helps Toxoplasma to survive for a long time in a form of cysts, evading clearance by the immune system. The occasional rapture of individual cysts is considered to be responsible for the continuous stimulation of the immune response. In immunocompromised patients toxoplasmosis is a life threatening condition. Interestingly, a rapture of a cyst followed by the dissemination of parasites causes more severe disease than a primary infection in this patient group. The most frequently involved organs are the brain, lungs and heart.

The biggest problem that toxoplasmosis poses in humans is vertical transmission of the parasite during pregnancy. Primary infection during pregnancy causes serious complications, and outcomes depend on the gestational age when infection occurs. In the first trimester, the risk of transmission of the parasite to the foetus is only 10%, but the outcomes are most severe. Most pregnancies would terminate and those remaining are associated with life threatening malformations of the foetus. In later gestational stages transmission of the parasite to the foetus occurs in up to 70% of cases, but causes relatively milder pathologies. Infected neonates are born with abnormalities of the brain and eyes and hepatic disorders, and are diagnosed with mental retardation later in life. Certain hygienic measures serve to prevent cases of congenital toxoplasmosis.

LITERATURE

  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. Harrington AM et al. (2008): Iron deficiency anemia, β-thalassemia minor, and anemia of chronic disease. Am J Clin Pathol, 129:466-471
  5. Baumgart DC, Sandborn WJ (2012): Crohn’s disease. Lancet, 3(80):1590-1605
  6. Thomas C et al. (2006): The diagnostic plot: a concept for identifying different states of iron deficiency and monitoring the response to epoetin therapy. Med Oncol. 23(1):23-26
  7. Thomas C and Thomas L (2005): Anaemia of chronic disease: pathophysiology and laboratory diagnostic. Lab Hematol. 11(1):14-23
  8. Danise P and Amendola G (2009): Nucleated red blood cells and soluble transferrin receptor in thalassemia syndromes: relationship with global and ineffective erythropoiesis. Clin Chem Lab Med. 47(12):1539-42
  9. Urrechaga E et al. (2011): Erythrocyte parameters in iron deficiency and thalassemia. J Clin Lab Anal. 25:223-228
  10. Robert-Gangneux F and Dardé ML (2012): Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev. 25(2):264-96

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