Reticulocyte haemoglobin content in the differential diagnosis of iron deficiency anaemia and thalassemia traits in pregnancy

Background: Iron deficiency anaemia (IDA) and thalassemia traits are the common conditions of microcytic hypochromic anaemia in pregnant women. However, the laboratory tests to differentiate them are expensive. The reticulocyte haemoglobin (Ret-Hb) test is relatively cheap. This study aimed to assess whether Ret-Hb can differentiate IDA from thalassemia traits in pregnant patients. Method: This cross-sectional study was conducted in the Bangabandhu Sheikh Mujib Medical University (BSMMU) from March 2023 to February 2024. We recruited pregnant women aged 18 to 40 through antenatal visits in the Obstetrics and Gynecology Department. Microcytic hypochromic anaemia was diagnosed by complete blood counts, IDA by iron profile and thalassemia traits by haemoglobin electrophoresis. Ret-Hb was measured using the flow cytometric method. Ninety pregnant wom-en—30 each with IDA, thalassemia traits and healthy individuals—were enrolled. Result: The mean age was 27 years. The IDA patients had significantly ( P <0.001) lower levels of Ret-Hb (mean 18.1, standard deviation 3.2 pg) compared to thalassemia traits (20.8, 2.2 pg) and healthy pregnant women (29.2, 1.9 pg). Using a Ret-Hb cut -off point of 19 pg, the test had 86.7% sensitivity and 53.3% specificity to differentiate thalassemia traits from IDA. Conclusion: Ret-Hb could be considered a diagnostic test to differentiate thalassemia traits from IDA in clinical settings before expensive confirmatory tests are performed.

populations (0.9%), and northern Europeans (0.1%). 8 In Bangladesh, total carriers of ß-globin gene mutations are 11.9%.Among them, 8.7% are HbE traits, and 2.2% are beta-thalassemia traits. 9During pregnancy, women with these traits usually present with more severe anaemia, which requires close maternal and fetal surveillance. 10e complete blood count can detect anaemia.IDA is confirmed by evaluating the iron profile.Thalassemia traits is diagnosed by Hb electrophoresis. 11Differential diagnosis of hypochromia is important clinically because treatment and prognosis depend on it.IDA is managed with iron supplementation.Iron overload can occur in the case of thalassemia traits. 4The confirmatory diagnostic assays for IDA and thalassemia traits are expensive, and the process is complex, which is sometimes difficult for a country with low resources. 12ore cost-effective and reliable screening tests are required.
Reticulocyte haemoglobin (Ret-Hb) content is a relatively new index that can help us understand marrow erythropoietic activity.Therefore, it is useful in evaluating microcytosis and hypochromia. 13Moreover, it is effective in evaluating a large number of samples. 14his study aimed to assess whether Ret-Hb can differentiate IDA and thalassemia traits in pregnant women.

Study design
This cross-sectional study was conducted from March 2023 to February 2024 in the Department of Laboratory Medicine in collaboration with the Department of Hematology and, Obstetrics and Gynecology of Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka.

Sample and sampling of subjects
We estimated the sample size of 30 for each group using the following formula 15 Where, µ1 (Mean in group I)=13.8,µ0 (Mean in group II)=11.9 4 , σ1 (Standard deviation in group 1)= 2.5, σ0 (Standard deviation in group ΙΙ)=2.5, u= 1.96 for 1% level of significance, v=0.84 (from Z table) at 80% power of the test.Therefore, we recruited 90 pregnant women (30 for each group).
We examined a total of 100 pregnant women consecutively.We included pregnant patients aged 18 to 40 through antenatal visits to the in-and out-patients.
IDA was confirmed by doing an iron profile (iron, ferritin, transferrin saturation, and total iron binding capacity).The thalassemia traits were confirmed by haemoglobin electrophoresis.Patients with IDA comprised 30 in number, and 30 patients with thalassemia traits were included.
Patients taking iron therapy, anaemia from chronic disorders, macrocytic anaemia, hereditary hemolytic anaemia other than thalassemia traits, history of blood transfusion within 30 days, and known cases of haematological malignancy were excluded from the study.Thirty healthy pregnant women were also recruited from the same setting.
Ethical standards of the Helsinki Declaration were maintained.All the participants were informed about the study objective, possible discomfort during venipuncture, and the study's benefits.Finally, written consent was obtained from all of them.

Measurement of Ret-Hb
Six mL blood was collected from the antecubital vein with aseptic precaution and separated into three aliquots: (a). 2 mL was taken into two ethylene diamine tetra-acetic acid tubes for analysis of Hb electrophoresis and CBC, and Ret-Hb, and (b) 2 mL into a plain tube for the iron profile.The tubes were labelled with the participant's identification number.All the laboratory tests were done on the day of blood collection.Reticulocyte haemoglobin iron deficiency anaemia and thalassemia trait in pregnancy 2 of 5 HIGHLIGHTS 1.Few studies have been done on pregnant anaemic patients to differentiate thalassemia traits from iron deficiency anaemia using reticulocyte haemoglobin content in Bangladesh.
2. Reticulocyte haemoglobin level of <19 pg showed high sensitivity but moderate specificity.
3. Reticulocyte haemoglobin content can differentiate thalassemia traits, but false negativity has to be kept in mind.A receiver operating characteristic curve analysis was performed, plotting sensitivity and 1-specificity values, to determine the area under the curve.P<0.05 was considered statistically significant.

RESULTS
The mean (standard deviation) age of the participants was 27 (4) years, with negligible difference between groups (P=0.89).As shown in Reticulocyte haemoglobin iron deficiency anaemia and thalassemia trait in pregnancy 3 of 5

DISCUSSION
Anaemia in pregnancy can have adverse maternal and fetal outcomes. 16Its early detection and differential diagnosis can prevent complications from anaemia and the treatment itself.We report that the Ret-Hb levels can differentiate IDA from thalassemia traits and healthy pregnant women with reasonable sensitivity and moderate specificity.Reticulocytes are immature, non-nucleated erythrocytes that contain ribosomal RNA residues.They mature three days after being produced in the bone marrow, while in circulation, maturity occurs after one day of release. 17alassemia includes α and β thalassemia traits caused by the defective synthesis of α and β chains in haemoglobin, respectively. 18Thalassemia is classified as major, intermedia, minor, or traits.The classification is done according to the impaired globin chain synthesis, resulting in variable phenotypes ranging from severe anaemia to clinically asymptomatic individuals. 19r findings are consistent with those of Düzenli Kar et al., 9 who found that Ret-Hb was significantly lower in patients with IDA than those with thalassemia traits.
The Ret-Hb level is a direct assessment of the incorporation of iron in Hb.In patients with IDA, the reason for low Ret-Hb is reduced iron storage.On the contrary, the iron storage in thalassemia traits remains unaffected. 20an et al. 21performed a receiver operating characteristics curve analysis for the differential diagnosis of IDA and beta thalassemia traits.They mentioned that when 19.1 pg was considered the Ret-Hb cutoff value, the area under the curve was 0.714 with a specificity of 84.1% and a sensitivity of 68.4%.This is similar to our findings.However, there is a lack of consensus about the cut-off point of Ret-Hb.Many authors used different cut-off points.For instance, Düzenli Kar 4 used a cut-off value <18.2 pg.Their sensitivity (65.3%) and specificity (96.9%) were a little different, but the area under the curve was almost similar (0.765). 4 Padang et al. 8 determined the cut-off value for Ret-Hb in differentiating the cases of iron deficiency anaemia with thalassemia traits of 27.8 pg with a sensitivity of 90.5% and a specificity of 71.4%.

Conclusion
We acknowledge the limitation of the findings based on purposively selected subjects.The reticulocyte indices can be used as a preliminary diagnostic tool to diagnose and differentiate IDA from thalassemia traits in Bangladeshi pregnant women attending tertiary care hospitals before expensive tests are considered.
Islam N et al. |Bangabandhu Sheikh Mujib Medical University Journal| 2024;17(3):e74482 The reticulocyte indices were measured by Automated Full Digital Cell Counter (Abbott Alinity HQ, Abbott Laboratories, USA) using flow cytometric methods in the Department of Laboratory Medicine, BSMMU.Complete blood count included estimation of Hb, mean corpuscular volume, mean corpuscular Hb, and red cell distribution width coefficient of variation.The flow cytometry methods used fluorescent staining of reticulocytes.The intensity of fluorescence was proportional to the RNA content of reticulocytes.Young, immature or stress reticulocytes had higher fluorescence than mature reticulocytes.The reticulocyte indices were derived from the reticulocyte scattergram generated by the forward light signals.Ret-Hb was the product of the reticulocyte volume and haemoglobin concentration determined by measuring individual reticulocytes' light scatter.Statistical analysisData were analysed using SPSS version 26.The mean (standard deviation) values were calculated for continuous variables, and frequency (per cent) was presented for the quantitative variables.The ANOVA (or Kruskal-Wallis) test, followed by the Tukey test, was used to compare continuous variables between IDA and thalassemia traits.The chi-square test was used to compare categorical data.Sensitivity and specificity were calculated to examine the performance of Ret-Hb<19 pg to differentiate IDA from thalassemia traits.

TABLE 1 Mean (standard deviation) of haematological indices including reticulocyte haemoglobin in pregnant women with iron deficiency anae- mia, thalassemia traits and healthy individuals (n=90)
A statistically significant difference between a: IDA and healthy; b: thalassemia traits and healthy women; c: IDA and healthy women; d: IDA and thalassemia traits.