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Original article
Reticulocyte parameters of delta beta thalassaemia trait, beta thalassaemia trait and iron deficiency anaemia
  1. Diego Velasco-Rodríguez1,2,3,
  2. Juan-Manuel Alonso-Domínguez1,
  3. Fernando-Ataúlfo González-Fernández1,4,
  4. Jesús Villarrubia1,3,
  5. María Sopeña1,
  6. Lorena Abalo1,
  7. Paloma Ropero4,
  8. Jorge Martínez-Nieto4,
  9. Félix de la Fuente Gonzalo4,
  10. Fernando Cava1
  1. 1Department of Haematology, Laboratorio Central de la Comunidad de Madrid, Madrid, Spain
  2. 2Programa de Doctorado de Investigación en Ciencias Médico-Quirúrgicas, Universidad Complutense de Madrid, Madrid, Spain
  3. 3Department of Haematology, Hospital Ramón y Cajal, Madrid, Spain
  4. 4Department of Haematology, Hospital Clínico San Carlos, Madrid, Spain
  1. Correspondence to Diego Velasco-Rodríguez, Department of Haematology, Central Laboratory of Madrid, Hospital Infanta Sofía, Paseo de Europa 34, 28702 San Sebastián de los Reyes, Madrid, Spain; diegovelascorodriguez{at}gmail.com

Abstract

Aims To analyse the differences in reticulocyte indices between delta beta thalassaemia trait (δβ-TT), beta thalassaemia trait (β-TT) and iron deficiency anaemia (IDA), and to correlate those differences with the physiopathological features of these three types of microcytoses.

Methods We performed a descriptive study of 428 samples (43 δβ-TT, 179 β-TT and 206 IDA) that were run on Advia 2120 analyser (Siemens). The following reticulocyte indices were assessed: absolute reticulocyte count (ARC), percentage of reticulocytes, mean corpuscular volume of reticulocytes (MCVr), haemoglobin content of reticulocytes (CHr), mean corpuscular haemoglobin concentration of reticulocytes, red blood cell distribution width of reticulocytes (RDWr), haemoglobin distribution width of reticulocytes (HDWr) and reticulocyte subpopulations based on their fluorescence according to mRNA (low (L-R), medium (M-R) and high (H-R)), MCV ratio and MCHC ratio. Correlation between fetal haemoglobin (HbF) and RDWr in patients with thalassaemia was evaluated.

Results RDWr was significantly higher in δβ-TT compared with β-TT (15.03% vs 13.82%, p<0.001), and so were HDWr (3.65% vs 3.27%, p<0.001), CHr (23.68 vs 22.66 pg, p<0.001) and MCVr (88.3 vs 85.5 fL, p<0.001). A good correlation was observed between HbF and RDWr (r=0.551, p<0.001). IDA subjects have more immature reticulocytes, but less ARC than β-TT, suggesting a certain degree of inefficient erythropoiesis in IDA in comparison with β-TT.

Conclusions Previously described differences between δβ-TT, β-TT and IDA in the corpuscular indices of mature red blood cell can also be observed in reticulocytes. The degree of anisocytosis in reticulocytes from patients with thalassaemia is correlated with HbF.

  • THALASSAEMIA
  • AUTOMATION
  • RETICULOCYTE COUNTS
  • IRON
  • LABORATORY TESTS

https://doi.org/10.1136/jclinpath-2015-203034

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    Introduction

    The study of reticulocytes has acquired great interest and increasing importance following the introduction of analysers that use specific dyes for RNA, since precise and accurate counts can be performed even at low concentrations.1 Reticulocytes are the earliest erythrocytes released into the bloodstream, and they circulate for only 1 or 2 days.2 Because of their shorter life span in the circulation compared with mature red blood cells (RBCs; 120 days), the reticulocyte parameters are more reliable tools to evaluate the bone marrow erythroid activity in real time, given that changes in erythropoiesis can be detected earlier.1

    Some reticulocyte parameters have proved to play a promising role in the study of several RBC disorders. For example, reticulocyte haemoglobin content (CHr) has been used to diagnose and follow-up iron deficiency anaemia (IDA),3 to predict early functional iron deficiency in healthy subjects,4 to discriminate between IDA and anaemia of chronic diseases5 and to distinguish thalassaemia carriers from healthy6–9 and iron-deficient subjects.1 ,10–19 Quantitative information about immature reticulocytes based on their amount of RNA can also be obtained, and maturation parameters may be helpful in the differential diagnosis of anaemias.17

    Iron deficiency and thalassaemia are the most common causes of microcytic anaemia, and their clinical management is quite different; so, the discrimination between thalassaemic and non-thalassaemic microcytosis has important implications.20

    Delta beta thalassaemia trait (δβ-TT) results from the deletion of beta and delta genes, and it is characterised by elevation of fetal haemoglobin (HbF) with normal values of HbA2.21 Patients with heterozygous condition are asymptomatic or develop mild anaemia, whereas homozygotes usually have thalassaemia intermedia. Most molecular mechanisms of beta thalassaemia are point mutations (single-base substitution) or insertions or deletions involving several nucleotides, while gene deletion is less common.21 Despite being less frequent than beta thalassaemia trait (β-TT), δβ-TT is not a rare condition in some geographical areas, and may be sometimes misdiagnosed; so, it is crucial to have efficient tools to distinguish both pathologies in order to give the patients a proper genetic counselling.

    The role of laboratory parameters of mature cells in δβ-TT has been explored by many authors.22–30

    Since reticulocytes are the previous stage in erythropoiesis, it seems reasonable to hypothesise that the differences in corpuscular indices of mature RBC could also be found in reticulocytes, and thus, contribute to explain the differences in the physiopathology of both entities.

    Although many studies have explored the utility of reticulocytes in thalassaemic carriers,1 ,6–19 none of them included patients with δβ-TT. The aims of our study were: (1) to analyse the differences in reticulocyte indices between δβ-TT, β-TT and IDA and (2) to correlate those differences with the physiopathological features of the three types of microcytosis.

    Materials and methods

    Study samples

    Over a 13-month period (March 2012–April 2013), 552 cases of microcytosis (MCV <80 fL) recruited in Central Laboratory of Madrid were initially included in the study: 74 δβ-TT, 272 β-TT and 206 IDA. Samples were all collected in K3-EDTA anticoagulant (Vacuette; Greiner Bio-One, Alphen aan de Rijn, The Netherlands) and a complete blood count was performed in all of them. Iron panel (serum iron, ferritin, transferrin and transferrin saturation index (TSI)) was performed in all of them. IDA subjects with Hb levels below 90 g/L were not included because they are not confused with thalassaemia trait in daily practice. In order to avoid possible interferences in the analysed parameters in patients with thalassaemia due to concomitant IDA, patients with thalassaemia and concomitant iron deficiency (ferritin <20 ng/mL and/or TSI <20%) were not included in the analysis. No other exclusion criteria were applied.

    Haematological analyses

    The reticulocyte analyses were all performed in the Advia 2120i analyser (Siemens Medical Solutions Diagnostics, Tarrytown, New York, USA) in a single run within 6 h of collection. The reticulocyte reagent causes isovolumetric sphering of RBCs to eliminate the variability of cell shape, and the reticulocytes are stained with a nucleic acid-binding dye called oxazine 750. Low-angle scatter (2°–3°), high-angle scatter (5°–15°) and absorbance are measured simultaneously by three detectors as the cells pass through the flow cell. Thus three different cytograms are generated by this technology: (1) high-angle scatter versus absorption, (2) low-angle scatter versus high-angle scatter (Mie cytogram or RBC map) and (3) volume versus Hb concentration. Quantitation and separation of reticulocytes are carried out by the absorption cytogram, and the amount of oxazine staining provides an additional subdivision into low-absorbing, medium-absorbing and high-absorbing cells.

    Serum ferritin and transferrin determination

    Ferritin, transferrin and TSI were measured by chemiluminescence immunoassay in the Advia Centaur (Siemens Medical Solutions Diagnostics, Tarrytown, New York, USA).

    Molecular analyses

    HbA2 and HbF levels were determined by high-performance liquid chromatography in the HA-8160 analyser (Menarini Diagnostics, Florence, Italy). Patients with increased HbA2 levels (>3.4%) were considered to have β-TT, whereas diagnosis of δβ-TT was made if HbF >3% and HbA2 ≤3.4%. In doubtful cases, multiplex ligation-dependent probe amplification (MLPA) study was carried out to screen for deletions in the human beta globin gene cluster. The MLPA is a comparative method based on the quantitative amplification and a subsequent fragment analysis of multiple probes hybridised across a region of interest.31 Since probe amplification can be achieved only when target DNA is in the sample, this method allows for a genetic profile showing the copy number variation of those targets in a patient's genome. Here, we used a commercial kit (MLPA kit P102-B2 HBB; MRC-Holland, Amsterdam, The Netherlands) that contains 28 probes designed to detect copy number changes in the haemoglobin beta locus, from 1 Mb upstream the locus control region to 10 Kb downstream of beta globin gene. MLPA reactions were performed according to the manufacturer's instructions and as previously described.31 Amplification products were separated by capillary electrophoresis on an ABI PRISM 3100 sequencer (Applied Biosystems, Foster City, California, USA). GeneMapper V.3.7 (Applied Biosystems) was used for size calling, and the data obtained were analysed with Coffalyser software (MRC-Holland). Five normal DNA samples, with normal RBC indices, were used as healthy controls for MLPA reactions.

    Reticulocyte parameters

    The following reticulocyte parameters obtained from the Advia 2120i analyser were assessed in the three groups of patients: absolute reticulocyte count (ARC), percentage of reticulocytes (%R), mean corpuscular volume of reticulocytes (MCVr), CHr, mean corpuscular haemoglobin concentration of reticulocytes (MCHCr), red cell distribution width of reticulocytes (RDWr), haemoglobin distribution width of reticulocytes (HDWr) and reticulocyte subpopulations based on their fluorescence according to mRNA (low (L-R), medium (M-R) and high (H-R)). Immature reticulocyte count (IR) was defined as the addition of M-R and H-R. The ratio of MCVr to MCV (MCV ratio) and the ratio of MCHCr to MCHC (MCHC ratio) were calculated to quantify the differences between reticulocytes and mature RBC in size and haemoglobin concentration, respectively.

    To analyse the impact of HbF in the RDWr, three subsets of patients were considered: β-TT with HbF <2%, β-TT with HbF >2% and δβ-TT.

    Statistical analysis

    Mean values and SD were the descriptive statistics used. Shapiro–Wilk test was used to assess the normality of the variables. Independent sample t test was used to compare reticulocyte indices between β-TT, IDA and δβ-TT. Bonferroni correction was used to counteract the problem of multiple comparisons, and p values <0.05 were considered to be statistically significant. Pearson coefficient was estimated to assess the correlation between HbF and RDWr, and one-way analysis of variance test was performed to compare RDWr between β-TT with HbF <2%, β-TT with HbF ≥2% and δβ-TT. One additional one-way analysis of variance test was performed to compare CHr between the three subsets of patients (β-TT, IDA and δβ-TT). The statistical software package SPSS V.19.0 for Windows was used for statistical analysis of the results (SPSS, Chicago, Illinois, USA).

    Results

    A total number of 43 δβ-TT, 179 β-TT and 206 IDA were finally evaluated. Mean values and SD of reticulocyte parameters of the three groups of patients are summarised in table 1.

    View this table:
    Table 1

    Reticulocyte parameters (mean±SD) in δβ-TT, β-TT and IDA

    No significant differences in ARC and %R were found between δβ-TT and β-TT. The reticulocytes in δβ-TT subjects presented significantly higher MCVr (88.30 vs 85.50 fL, p<0.001), higher CHr (23.68 vs 22.66 pg, p<0.001), higher RDWr (15.03% vs 13.82%, p<0.001) and higher HDWr (3.65% vs 3.27%, p<0.001).

    Differences in RDWr values in β-TT with HbF <2% (13.52%), β-TT with HbF ≥2% (14.79%) and δβ-TT (15.03%) were statistically significant (p<0.001) (figure 1). Pearson coefficient showed good correlation between the percentage of HbF and RDWr (r=0.551, p<0.001) (figure 2).

    Figure 1
    Figure 1

    Box-and-whisker plot showing red blood cell distribution width of reticulocytes (RDWr) values in betathalassaemia trait (β-TT) with fetal haemoglobin (HbF) <2%, β-TT with HbF >2% and delta beta thalassaemia trait. Differences were statistically significant (p<0.001).

    Figure 2
    Figure 2

    Relationship between the percentage of fetal haemoglobin (HbF) and red blood cell distribution width of reticulocytes (RDWr). Correlation coefficient was calculated by Pearson method (r=0.551, p<0.001).

    None of the reticulocyte subpopulations based on RNA content showed significant differences between δβ-TT and β-TT.

    In β-TT, ARC was higher (96.49×109/L) than in IDA subjects (87.07×109/L, p<0.001) as well as %R (1.91% vs 1.71%, p=0.005), MCHCr (26.70 vs 25.39 g/dL, p<0.001) and RDWr (13.82% vs 12.80%, p<0.001). The size of the reticulocytes (85.50 vs 94.95 fL, p<0.001) and the haemoglobin content (22.66 vs 24.05 pg, p<0.001) were lower in β-TT carriers. Significant differences in some of the reticulocyte subpopulations were also found: %L-R (81.96% vs 78.05%, p<0.001), L-R (718.25×109/L vs 552.36×109/L, p<0.001), %M-R (14.67% vs 16.31%, p<0.001), %H-R (3.36% vs 6.02%, p<0.001) and %IR (18.03% vs 22.33%, p<0.001).

    MCVr was higher in IDA (94.95 vs 88.30 fL, p<0.001) than in δβ-TT, whereas MCHCr (25.39 vs 26.93 g/dL, p<0.001), RDWr (12.80% vs 15.03%, p<0.001) and HDWr (3.15% vs 3.65%, p<0.001) were higher in δβ-TT. H-R was the only maturation parameter that showed significant differences between these two entities, being higher in IDA (41.29×109/L vs 26.05×109/L, p<0.001).

    Differences in CHr values in β-TT (22.66 pg), δβ-TT (23.68 pg) and IDA (24.05 pg) were statistically significant (p<0.001) (figure 3).

    Figure 3
    Figure 3

    Box-and-whisker plot showing reticulocyte haemoglobin content (CHr) values in delta beta thalassaemia trait, betathalassaemia trait and iron deficiency anaemia. Differences were statistically significant (p<0.001).

    MCV ratio was >1, and MCHC ratio was <1 in the three groups, with no significant differences between them.

    Discussion

    Determination of the number of reticulocytes and some of their cellular properties may provide reliable information about the bone marrow activity.32 Modern automated cell counters produce more accurate results than the manual method and provide information about reticulocyte immaturity, due to the use of fluorescent stains that bind to ribosomal RNA.17 There is great variability across the different analytical platforms in the additional parameters generated by automated analysers; thus, it is difficult to validate them as diagnostic tools. However, reticulocyte indices provide valuable information for the understanding of the physiopathology of several RBC disorders.

    δβ-TT versus β-TT

    Laboratory findings in δβ-TT and β-TT are quite similar. However, differences in several parameters of mature RBC between both entities have been reported by many authors so far.22–30 RDW was the only parameter that showed significant differences between both types of thalassaemia in all of them, being more elevated in δβ-TT. MCV26 ,29 ,30 and MCHC26 ,30 of mature RBC are higher in patients with δβ-TT. In a recent study, RDW demonstrated to be strongly affected by the percentage and the absolute value of HbF in patients with β-TT and δβ-TT.30 Moreover, a threshold of 17.35% for RDW identified 90.2% of δβ-TT and 84.9% of β-TT.30 However, none of the mentioned studies included reticulocyte indices.

    We decided to evaluate reticulocyte parameters in these two conditions to see if differences in corpuscular indices of mature RBC can also be found in reticulocytes, since reticulocytes are the previous stage in erythropoiesis.

    Reticulocytes of δβ-TT have more anisocytosis than β-TT, probably due to the heterogeneous distribution of HbF in erythroid precursors, which can be demonstrated with the Kleihauer–Betke test, although the reasons for this fact remain unknown.33 Differences in RDWr were found even among patients with β-TT with and without elevation of HbF (figure 1). The results of this study demonstrate that RDWr is influenced by the percentage of HbF in patients with β-TT and δβ-TT. This finding is in agreement with a recently published report where the same correlation was observed between HbF and RDW of mature RBC.30 However, the correlation in reticulocytes appears to be weaker. The increase of HbF in δβ-TT subjects is due to an overexpression of gamma globin genes by a mechanism of loss of competence of transcription factors that regulate the expression of beta globin genes.34 As a consequence of delta and beta genes’ deletion, those transcription factors interact with the gamma-locus promoter zones. Phenotypic expression of HbF depends on the size and location of the deleted sequences. Loss of regulatory regions of the expression of gamma globin genes can also influence HbF synthesis.35

    Microcytosis in patients with thalassaemia can be explained by a higher number of divisions in erythroid precursors due to impaired globin chain synthesis and subsequent decreased haemoglobinisation.30 In δβ-TT subjects, there is an increase in gamma chain synthesis that may partially compensate for the lack of beta globin chain synthesis.30 Therefore, haemoglobinisation of δβ-TT erythroid precursors may be increased compared with β-TT, which would explain their higher CHr (23.68 vs 22.66 pg, p<0.001), and they may consequently undergo fewer divisions and have higher MCVr (88.30 vs 85.50 fL, p<0.001) than β-TT subjects.

    CHr is the product of the haemoglobin concentration and the cell volume. When the reticulocytes mature into erythrocytes, the haemoglobin concentration increases as the cell volume decreases; thus, CHr seems to be a more stable parameter than MCHCr.3 Although differences in MCHCr were not significant, probably due to an insufficient δβ-TT sample size, subjects with δβ-TT showed higher MCHCr than β-TT subjects (26.93 vs 26.70 g/dL). Although CHr has proved to distinguish thalassaemia carriers from healthy6–9 and iron-deficient subjects,1 ,10–19 it has not, however, been evaluated to date in δβ-TT. Skarmoutsou et al6 demonstrated that significant differences in CHr were even found between silent β-TT, β+-TT and β0-TT. CHr was in correlation with the degree of globin chain imbalance, being β0-TT the group with the lowest values. Analysis in different types of β-TT was not carried out in our study.

    Immature reticulocytes are released from the bone marrow when erythropoietic activity is enhanced.36 In our study, none of the immature reticulocyte subpopulations according to their RNA content showed significant differences between δβ-TT and β-TT.

    β-TT versus IDA

    Patients with β-TT usually present with higher MCHC and lower MCV and RDW in mature RBC compared with patients with IDA.28 ,37–40 As expected, mean MCVr was more decreased in β-TT (85.50 fL) than in IDA (94.95 fL, p<0.001), and the MCHCr showed an opposite trend (26.70 vs 25.39 g/dL, p<0.001). The unbalanced chain synthesis and the excess of free alpha chains lead to membrane oxidant damage, potassium loss and relative dehydration in β-TT subjects. Iron plays a key role in DNA synthesis, being part of ribonucleotide reductase, which catalyses the formation of deoxyribonucleotides.41 Therefore, iron deficiency leads to fewer divisions of erythroid precursors and to a certain degree of macrocytosis compared with β-TT. Although MCHCr was lower in IDA, CHr was higher in IDA compared with β-TT subjects (24.05 vs 22.66 pg, p<0.001). Thus, CHr seems to be influenced by haemoglobinisation and especially by the size of RBCs. These results are similar to those reported previously by Vicinanza et al,7 Chouliaras et al,13 Urrechaga et al18 and Ceylan et al19 using the same analytical platform. However, some of them reported insufficient sensitivity and/or specificity of CHr for discriminating both entities.13 ,19 CHr values should be interpreted in the context of the overall erythrocyte physiology of the patient due to its diagnostic limitations in patients with recent blood transfusions, iron therapy or concomitant vitamin B12 or folate deficiency.3 Vicinanza et al7 proposed ΔCHr as a more precise parameter than CHr, since CHr seems to be less efficient to discriminate patients with pronounced macrocytosis or microcytosis.4 ΔCHr can be calculated as the difference between measured CHr and the CHr expected for the corresponding MCVr (ΔCHr=CHr−CHr-e). Sysmex analysers (Sysmex, Kobe, Japan) provide a parameter called reticulocyte haemoglobin equivalent (Ret He, also known as RET-Y), similar but not identical to CHr, that has also been evaluated in β-TT and IDA.1 ,11 ,12 ,14 ,16 Whereas Noronha and Grotto11 and Bartels et al 12 found no significant differences between both pathologies, other authors reported differences between β-TT and mild14 or all types of IDA,1 and Sudmann et al16 evaluated an efficient and easy-to-calculate algorithm, including Ret He, RBC and ferritin to discriminate between β-TT and IDA.

    It may seem surprising that RDWr was higher in β-TT (13.82% vs 12.80%, p<0.001) than in IDA, contrary to what happens in mature RBC, which shows higher degree of anisocytosis in IDA subjects.39–42 RDWr is the coefficient of variation of MCVr, defined as the ratio of its SD to its mean. As the mean MCVr (the denominator of the formula) of patients with IDA is higher, their RDWr is lower. Moreover, the cell size of reticulocytes is bigger than mature RBC; so, the RDWr is slightly lower than RDW in both pathologies. In this setting, SD seems to be a more precise measure of dispersion, being more elevated in patients with IDA (1.45 vs 1.37).

    IDA subjects had more immature reticulocytes than β-TT, suggesting a more enhanced erythropoietic activity, similar to the results reported by Noronha and Grotto11 and Urrechaga et al.14 ,18 However, consistent with previous reports,11 ,12 ,14 the absolute number of reticulocytes was lower in patients with IDA. Therefore, they have more immature reticulocytes, but many of them do not reach the bloodstream, probably suggesting a certain degree of inefficient erythropoiesis in IDA in comparison with β-TT. The higher proportion of immature reticulocytes in IDA compared with thalassaemic subjects may be hypothetically a consequence of a higher expression of soluble transferrin receptor (sTfR) mRNA.42 In response to insufficient supply of transferrin iron, the synthesis of sTfR is increased proportionally to the cell's iron requirement.43 Therefore, an increase of sTfR plasma levels may reflect an increase in erythroid precursors.6 The sTfR has also proved to be correlated with HbA2 (which reflects the degree of globin chain imbalance) in patients with β-TT.6 However, De Lima and Grotto36 found no significant differences in sTfR and immature reticulocytes between β-TT and IDA. Unfortunately, we could not evaluate sTfR in this study, since its determination is not available in our laboratory.

    δβ-TT versus IDA

    Corpuscular indices were all significantly different between IDA and δβ-TT with the exception of CHr. Reticulocytes of δβ-TT subjects were smaller (MCVr 88.30 fL) and denser (MCHCr 26.93 d/dL) than iron deficiency reticulocytes (MCVr 94.95 fL and MCHCr 25.39 g/dL, p<0.001). The heterogeneous distribution of HbF in erythroid precursors explains the higher degree of anisocytosis (RDWr 15.03% vs 12.80%, p<0.001) and anisochromia (HDWr 3.65% vs 3.15%, p<0.001) found in δβ-TT reticulocytes. The increase of haemoglobinisation of erythroid precursors in δβ-TT makes them undergo fewer divisions, so differences in MCVr can be explained. None of the maturation parameters showed significant differences between these two entities, with the exception of H-R. Reticulocytes with high mRNA content were much higher in patients with IDA (41.29×109/L vs 26.05×109/L, p<0.001), confirming that the expansion of the erythron is more increased in iron-deficient subjects. As it occurred with β-TT, the ARC in IDA was lower than in δβ-TT despite having more immature reticulocytes, suggesting a more ineffective erythropoiesis. Non-significant differences in the rest of maturity parameters could possibly be attributed to the relatively small δβ-TT sample size.

    Maturation ratios

    The MCVr was consistently higher than MCV in the three groups we have studied, whereas the behaviour of MCHCr and MCHC showed an opposite trend. Our results are in agreement with d’Onofrio et al10 and Ceylan et al,19 and confirm the classical concept that maturation of the reticulocytes is associated with increase in density and loss of size.

    Conclusions

    Previously described differences between δβ-TT and β-TT in the corpuscular indices of mature RBC can also be observed in reticulocytes. The size of δβ-TT reticulocytes is slightly bigger and their haemoglobin content is slightly higher in comparison with β-TT, and a higher degree of anisocytosis is also observed due to a heterogeneous distribution of HbF in erythroid precursors of δβ-TT. The most immature reticulocytes (with the highest RNA content) are higher in IDA subjects compared with thalassaemic individuals, probably reflecting both an enhanced erythropoiesis that seems to be even more inefficient than thalassaemic erythropoietic activity, and also a higher expression of the mRNA of sTfR. To our knowledge, this is the first study that evaluates reticulocyte parameters in δβ-TT subjects. A possible drawback in our study is the fact that molecular analysis was not performed in all the samples. Sequencing of the beta globin gene was only performed in those samples with non-conclusive percentages of HbA2 and HbF.

    Clinical applicability of most of these parameters is still to be determined, since the great majority of them are not validated, and no commercial QC material for each of them is available, thus, these results should be interpreted with caution.

    In conclusion, although the analysis of corpuscular indices and maturation parameters of reticulocytes cannot replace molecular diagnosis in haemoglobinopathies, it provides qualitative information about their cellular properties, and may contribute to the understanding of the physiopathology of these entities.

    Take home messages

    • Delta beta thalassaemia trait (δβ-TT) reticulocytes have slightly higher mean corpuscular volume of reticulocytes and haemoglobin content of reticulocytes than beta thalassaemia trait (β-TT) reticulocytes.

    • A higher degree of anisocytosis (red blood cell distribution width of reticulocytes) is also observed in δβ-TT reticulocytes in comparison with β-TT reticulocytes due to a heterogeneous distribution of fetal haemoglobin in erythroid precursors.

    • Iron deficiency anaemia subjects present a higher percentage of immature reticulocytes, but lower absolute number of reticulocytes compared with thalassaemic individuals, suggesting a more inefficient erythropoietic activity.

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    Footnotes

    • Handling editor Mary Frances McMullin

    • Contributors DV-R validated the complete blood counts, collected the data and wrote the article. J-MA-D validated the complete blood counts, made the statistical analysis and reviewed the manuscript. F-AG-F validated the HPLC analysis, helped in the interpretation of the results and reviewed the manuscript. JV helped in the interpretation of the results and reviewed the manuscript. MS and LA helped in the collection of the data and reviewed the manuscript. PR, JM-N and FdlFG did the molecular analysis of the doubtful thalassaemic samples. FC reviewed the manuscript.

    • Competing interests None.

    • Provenance and peer review Not commissioned; externally peer reviewed.