Sickle cell anaemia with iron deficiency

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. 

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).