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

Can you explain the abnormal RBC morphology of this girl? Beta-thalassaemia intermedia
Sickle cell anaemia with iron deficiency
Autoimmune haemolytic anaemia
Hereditary spherocytosis

Online version of this month´s case:

The correct answer to November´s quiz is:

Sickle cell anaemia with iron deficiency

Scattergrams and microscopy:

Patient history: a fatigued 4-year old girl was brought to the hospital.

 

Table:

Interpretation and differential diagnosis:

The answer can be inferred from…

  • Microcytosis                                                 low MCV and high MicroR%
  • Hypochromic anaemia                               low HGB, low MCH and high HYPO-He
  • High RBC size distribution                        high RDW-CV
  • Reticulocytosis                                             high RET# and RET%
  • Ineffective erythropoiesis                         low RET-He and Delta-He    
  • No extracellular haemoglobin                  No difference between HGB and HGB-O

 

Case history
A fatigued 4-year old girl was brought to the hospital. A blood sample was sent for an analysis.

Case results
The patient presented with hypochromic (low MCH, high HYPO-He) microcytic (low MCV, high HYPO-He) anaemia (low HGB) with reticulocytosis (high RET# and RET%). Chronic inefficient haemoglobinisation of the erythropoiesis (low RET-He and Delta-He) was also observed. Microcytes are present in iron deficiency anaemia (IDA), functional iron deficiency (FID), haemoglobinopathies such as thalassaemia, and hereditary spherocytosis (microcytosis without hypochromia). Microcytic anaemia of iron deficiency or functional iron deficiency and thalassaemia can be differentiated using the Urrechaga index - a formula that takes into account red blood cell parameters from the blood count (1-2):

Urrechaga index (MicroR% - HYPO-He% - RDW-CV%)
> -5.1 → β-thalassaemia
< -5.1 → iron deficiency

In this case, the Urrechaga index equals to -22,5, strongly suggestive of IDA or FID rather than β- thalassaemia. The total amount of microcytes (MicroR 47%) and the low MicroR/Hypo-He ratio (47/46,9 = 1) indicate an IDA status.

However the reticulocytosis with the ineffective haemoglobinisation (low RET-He and normal Delta-He) hints to a second underlying disease besides the IDA situation. Circulating NRBC and the markedly increased reticulocyte count indicate a haemolytical disease. The increased fragment or abnormal RBC shape count and the presence of sickle cells in the smear confirm the underlying sickle cell disease (SCD). SCD can also explain the elevated WBC count due to lymphocytosis, as this is a frequent observation in children with SCD (3).Haemoglobin electrophoresis and solubility test confirmed the result.

 

The following answers are incorrect for the described reasons

Beta-thalassaemia intermedia
β- thalassaemia is an inherited haemoglobinopathy characterized by hypochromic microcytic anaemia. It should be differentiated from other haemoglobinopathies and iron deficiency anaemia (IDA) (3).Moderate anaemia, hypochromic microcytes (high Micro-R and HYPO-He, low MCH, low MCV), reticulocytosis with ineffective haemoglobinisation (low RET-He), presence of erythroblasts (NRBC) in the blood smear can be indicative of β- thalassaemia. However, the number of NRBCs in this case is untypically low for thalassaemia intermedia. MicroR and HYPO-He are 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 inthalassaemia the ratio MicroR to HYPO-He is mostly increased (5). In this case, the ratio is equal to 1, indicative of iron deficiency condition. Apart from that, the Urrechaga index is equal to -22,5, strongly suggestive of iron IDA and excluding thalassaemia as a possible diagnosis of this patient.

Autoimmune haemolytic anaemia
Autoimmune haemolytic anaemia (AIHA) refers to a collection of disorders characterized by the presence of autoantibodies that bind to the patient's own erythrocytes, leading to premature red cell destruction. In all cases of AIHA, the autoantibody leads to a shortened red blood cell survival (ie, haemolysis) and, when the rate of haemolysis exceeds the ability of the bone marrow to replace the destroyed red cells, to anaemia and its attendant signs and symptoms.The pathophysiology of red cell destruction depends on the type of agglutination (6). In the cold-agglutinine AIHA, agglutination of RBC leads to discrepant results between RBC-I and RBC-O, which are counted in the RET-channel. The reason for it is the high temperature in the RET channel that leads to disintegration of the agglutinins, therefore the RBC-O count is more accurate than RBC-I in cases of cold-agglutinin AIHA. In case of this patient RBC-I and RBC-O showed no difference and no RBC agglutination was observed in the smear, which excludes the possibility of cold-agglutinin AIHA . The warm-reactive AIHA is an acute condition leading to intravascular haemolysis. The presence of intravascular haemolysis can be confirmed by discrepant results for HGB measured from the haemolysed sample by the SLS-method, and for HGB-O (research parameter), calculated based on parameters measured in the RET channel, and equivalent to intracellular haemoglobin. In this case there were no discrepancies between HGB (81 g/L) and HGB-O (80 g/L), which confirms the absence of extracellular haemoglobin and intravascular haemolysis.Warm-agglutinin AIHA coexisting with an IDA could lead to a microcytic anaemia with reticulocytosis. However, in such extreme situation the intravascular haemolysis would also lead to a difference between HGB and HGB-O (described above), thus this scenario can be excluded as a reason for the abnormal blood parameters in this patient. 

Hereditary spherocytosis
Hereditary spherocytosis (HS) is caused by a variety of molecular defects in the genes that code for the red blood cell membrane proteins. As a result, the erythrocytes have a shape of a sphere (spherocytes) instead of a biconcave shape. These abnormal red blood cells are sequestrated in the spleen. The lifespan of such RBCs is reduced from 120 to 10-30 days. Destruction of the spherocytes in the spleen leads to haemolysis and on a long run could lead to anaemia if not compensated by increased erythropoiesis (demonstrated by reticulocytosis). The typical blood analysis results show reticulocytosis and small volume of RBC. In the blood smear, RBCs appear abnormally small and lack the central pale area.For the differential diagnosis of haemolytic anaemias, several parameters are used in combination (7). A specific feature of the automated measurement is exploited in order to differentiate between causes of spherocytosis.HS defect of the erythrocyte membrane results in RBC membrane loss, with apparent reduction in appearance of cell size, but no loss of cellular content. On the XN these changes translate into an increase in the percentage of microcytic cells (elevated %MicroR), which is not matched by an equivalent increase in poorly haemoglobinised cells, i.e. no or only minimally elevated %HYPO-He.Furthermore, the membrane defect in HS results in a discrepancy between the detected high reticulocyte count (RET#) in comparison with a proportionally low immature reticulocyte fraction (IRF) of the XN. The observed result is likely to be caused by an insufficient entry of the fluorescence dye into the defective cells. Consequently the ratio between total reticulocytes and the immature reticulocyte fraction is abnormal which is specific for hereditary spherocytosis. In reticulocytosis due to a different cause, such as thalassaemia or any other haemolytic anaemia, an increased IRF fraction should be expected.Prominent anaemia, poor haemoglobinisation of reticulocytes (high HYPO-He) and high IRF% observed in this patient are not characteristic of HS. 

Underlying disease:

Sickle cell disease (SCD) is an inherited group of disorders characterized by the presence of haemoglobin S (HbS). The hallmarks of SCD are vasoocclusive phenomena and haemolytic anaemia (8).

Haemoglobin normally is soluble in the erythrocyte and does not polymerize. Haemoglobin S (HbS) is an abnormal haemoglobin that results from a point mutation in the beta globin gene that causes the substitution of a valine for glutamic acid as the sixth amino acid of the beta globin chain. The resulting haemoglobin tetramer becomes poorly soluble when deoxygenated.
The pathological polymerization of deoxygenated HbS is essential to vasoocclusive phenomena. The polymer assumes the form of an elongated ropelike fiber, which usually aligns with other fibers, resulting in distortion of erythrocytes into the classic crescent or sickle shape and a marked decrease in red cell deformability. However, polymerization alone does not account for the pathophysiology of SCD. Subsequent changes in red cell membrane structure and function, disordered cell volume control, and increased adherence to vascular endothelium also play an important role. 
The sickling is promoted by conditions which are associated with low oxygen levels, increased acidity, or low volume of the blood. These conditions can occur as a result of injury to the body's tissues, dehydration, or anaesthesia. The sickle red blood cells clump together and stick to the walls of blood vessels, blocking blood flow. Sickling of the cells can cause attacks of sudden, severe pain, called pain crises. Most children with SCD are pain free between painful crises, but adolescents and adults may also suffer with chronic ongoing pain. The red cell sickling and poor oxygen delivery can also cause permanent damage to the brain, heart, kidneys, liver, spleen, and bones. The severity and symptoms vary greatly from person to person, even within the same family. The lifespan of sickle cell red blood cells is reduced from 120 to 10-20 days. The cells tend to haemolyze, which when not compensated by efficient erythropoiesis, can lead to haemolytic anaemia over a course of time.
Clinical manifestations of SCD are not present at birth, and usually begin to become apparent after the first few months of life as the concentration of HbS rises and of fetal haemoglobin declines. Sickled cells can be seen in the peripheral blood of children with SCD at three months of age, and moderately severe haemolytic anaemia is apparent by four months of age.
Diagnosis of sickle cell disorders can be made with haemoglobin electrophoresis in cellulose acetate or citrate agar, isoelectric focusing, by high performance liquid chromatography (HPLC) and solubility tests, or direct DNA testing.The chronic haemolysis in those with sickle cell anaemia is usually associated with a mild to moderate anaemia (hematocrit 20 to 30 percent), reticulocytosis of 3 to 15 percent, accounting for elevated MCV, unconjugated hyperbilirubinemia, elevated serum LDH and low serum haptoglobin. The peripheral blood smear reveals sickled red cells, polychromasia indicative of reticulocytosis, and Howell-Jolly bodies. The red cells are normochromic unless there is coexistent thalassemia or iron deficiency. If the age-adjusted MCV is not elevated, the possibility of sickle cell-beta thalassemia, coincident alpha thalassemia, or iron deficiency should be considered. Mean white blood cell (WBC) counts are higher in patient with sickle cell anaemia than in normal population, particularly in those under the age of 10 (3).Sickle cell disease is most common in Africans and African-Americans. It is also found in other ethnic and racial groups, including people from South and Central America, the Caribbean, Mediterranean countries, and India.

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. 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
  3. West MS et al. (1992): Laboratory profile of sickle cell disease: a crosssectional analysis. The Cooperative Study of Sickle Cell Disease. J Clin Epidemiol, 45:893.
  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. Urrechaga E et al. (2011): Erythrocyte parameters in iron deficiency and thalassemia. J Clin Lab Anal. 25:223-228
  6. Gehrs BC et al. (2002): Autoimmune hemolytic anemia. Am J Hematol, 69:258
  7. Cynober T et al. (1996):  Red cell abnormalities in hereditary spherocytosis: relevance to diagnosis and understanding of the variable expression of clinical severity. J Lab Clin Med, 128:259
  8. Bunn HF (1997): Pathogenesis and treatment of sickle cell disease. N Engl J Med, 337:762

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