ROLE OF RETICULOCYTE HEMOGLOBIN CONTENT IN DIAGNOSIS OF IRON DEFICIENCY ANEMIA

Objectives : Anemia is a global problem of immense health significance affecting persons of all ages and economic groups. Iron deficiency anemia (IDA) is the most common type of anemia met with in clinical practice. For IDA diagnosis, estimation and treatment, many indices such as serum iron (SI), total iron binding capacity (TIBC), serum ferritin (SF), and soluble transferrin receptor assay are used. But reticulocyte hemoglobin content (CHr) is called as the gold standard for diagnosing IDA as it is the most valuable screening tool for identifying IDA with a sensitivity of 94% and specificity 80% and differentiates IDA from anemia of systemic disease. The present study was undertaken to evaluate CHr as a most efficient marker in diagnosing IDA. Methods: This prospective observational study was carried out in the Department of General Medicine of M.K.C.G. Medical College and hospital, Berhampur, Odisha, India, from October 2017 to October 2019. Sixty microcytic hypochromic patients of either sex >18 years of age admitted in the medicine ward fulfilling the inclusion and exclusion criteria were included in this study. After taking detailed history and clinical examination, laboratory investigations including complete blood count, SI, serum, ferritin, serum transferrin saturation, TIBC, CHr, and bone marrow aspiration with iron stain were done in all patients. Results: In the study group of 60 patients, 10 (16.66%) patients had mild anemia, 17 (28.33%) had moderate anemia, and 33 (55%) had severe anemia. Mean hemoglobin of the patients was 6.86 g/dL and SD was 1.95 g/dL. Nineteen (31.66% patients) had TIBC in the range of 351–400 µ g/dl. Mean±SD of serum TIBC was 333.91±67.26 µ g/dL. Thirty-nine patients (65%) had transferrin saturation in the range of 0.1–10%. The mean±SD of the study group was 13.68±3.22%. Fifty (83.33%) patients had SF in the range of 0–100 µ g/dL. Twenty-three patients (38.33%) had CHr concentration between 15.1 and 20 pg followed by 19 (31.66%) between 20.1 and 25 pg and 18 (30%) between 25.1 and 30 pg. The mean±SD of this study was 22.14 pg±3.92. Conclusion: CHr is found to be a potential biomarker that can be used to differentiate IDA from other causes of anemia.


INTRODUCTION
Anemia is defined as "Hemoglobin level in blood below the lower extreme of the normal range for the age and sex of individual" [1]. It is a global problem of immense health significance affecting persons of all ages and economic groups. It has been estimated that 20% of the world's population is iron deficient. It occurs at all ages, but is especially common in women of child bearing age, in whom it is an important cause of chronic fatigue and ill health [1]. During the reproductive life of the female, menstruation, pregnancy, parturition, and lactation the physiological requirements of iron increase significantly [1,2].
Iron deficiency anemia (IDA) is the most common type of anemia throughout the world and according to Idris et al., when iron deficiency is widespread and severe, the prevalence of morbidity and effects on the individual's resistance to infectious disease are significant [3].
The screening procedure used most often is blood hemoglobin determination. Values falling below the cutoff point are considered abnormal. The normal range of hemoglobin values for adult males is 13.5-17.5 g/dL and that for adult females is 12.5-15 g/dL. The World Health Organization (WHO) defines anemia as a hemoglobin level <13 g/dL in men, <12 g/dL in non-pregnant women, and <11 g/dL in pregnant women. Anemia in pregnancy is common. This is related to increased demands of iron during pregnancy, pre-existing negative iron balance due to frequent pregnancies, menstrual blood loss, dietary inadequacies, helminthiasis, and amoebiasis are important contributory factors [2,4,5]. In India and other developing countries, incidence of nutritional anemia in reproductive age groups ranges from 60% to 80% compared to 10-20% in developed countries [6]. In Asia, the prevalence of nutritional anemia is particularly high in countries such as Bangladesh (74-80%), Indonesia (37-73%), India (34-69%), and the Philippines (42-47%) [7].
For IDA diagnosis, estimation and treatment, many indices such as serum iron (SI), total iron binding capacity (TIBC), serum ferritin (SF), and soluble transferrin receptor assay are used. Gorden et al. have shown that SF is the best investigation for distinguishing those with iron deficiency from those who were not iron deficient. Appropriate use of SF would refute diagnosis of iron deficiency without a bone marrow aspirate in 70% of patients [8].
Reticulocyte hemoglobin content (CHr) is a measurement of hemoglobin inside the reticulocyte. It correlates directly with the functional availability of iron in bone marrow. Just like any other perfect laboratory test it is accurate, simple, and less expensive [9]. Reticulocytes are newly produced, relatively immature red blood cells. Reticulocyte count or percentage is an indicator of ability of persons bone marrow to produce adequate RBCs (erythropoiesis).

Pattanayak et al.
Reticulocytosis reflects responsive marrow. Reticulocytopenia suggests nonfunctional bone marrow. Automated reticulocyte counts have greater precision, accuracy and reproducibility than manual counts [10]. The reference range for reticulocyte count for adults is 0.5-1.5%. Corrected reticulocyte count (CRC) or reticulocyte index (RI) give more accurate assessment of marrow function. The reference range for CRC in adults is 0.5-1.5%. Modern automated particle cell counters utilize flow cytometry technique and measure reticulocyte cellular characteristics, that is, immature reticulocyte fraction (IRF) and CHr [11,12]. CHr is most valuable screening tool for identifying IDA with a sensitivity of 94% and specificity of 80% [13,14]. In anemia of systemic disease (ASD) functional iron deficiency may be seen because of poor absorption from GI tract of increased production of hepcidin which in turn trap Fe in reticuloendothelial system (RES). This condition (ASD) confuses with IDA. CHr differentiates IDA from ASD. The discriminatory power of CHr both with respect to sensitivity and specificity is better than mean corpuscular volume (MCV) and ferritin [11]. CHr predicts early response to treatment of IDA whereas Hematocrit, RBC indices take weeks to predict response to treatment.
Today CHr is called as the gold standard for diagnosing IDA replacing both of these. Despite its simplicity and utility, it is rarely used in clinical practice. In our state particularly southern Odisha IDA is very common problem which needs to be diagnosed early. Hence, CHr can be an extremely valuable recent addition for diagnosis and assessment of therapeutic response and also it differentiates from ASD which many time confuses with IDA.
The present study was undertaken to evaluate reticulocyte hemoglobin content (CHr) as a most efficient marker in diagnosing IDA.

MATERIALS AND METHODS
A prospective observational study was designed and conducted in the Department of General Medicine in MKCG Medical College and Hospital, Berhampur, Odisha, India, over a period of 2 years from October 2017 to October 2019. A total number of 60 microcytic hypochromic patients of either sex above the age of 18 years fulfilling the inclusion criteria were taken in this study. The study was conducted after the study protocol was approved by the Institutional Ethics Committee (No. 630/ Chairman-IEC, M.K.C.G. Medical College, Berhampur-4). Informed consent was obtained from all the patients and the study was done in accordance with the guidelines of the Declaration of Helsinki 2008.

Inclusion criteria
The following criteria were included in the study: • Patients with microcytic hypochromic anemia • >18 years of age • Patients agreed to undergo bone morrow aspiration for diagnosis.

Exclusion criteria
The following criteria were excluded from the study: • Pregnant women • Patients who had received blood transfusion, oral, or IV iron supplementation within a month • Patients suffering from any inflammatory disorders • Patients suffering from hemoglobinopathy, leukemia, or myelodysplastic syndrome (MDS) • Patients having mean MCV >80 fl.
All the 60 patients were subjected to detailed history and clinical examinations. They underwent the routine laboratory investigations such as complete blood count (CBC), comment on perepheral smear (CPs), SI, TIBC, SF, serum transferrin saturation, reticulocyte hemoglobin content (CHr), bone marrow iron estimation, C-reactive protein, erythrocyte sedimentation rate, serum electrolytes, renal function test, liver function test, FBS, PPBS, HBSAg, ICTC, and urine examination.
Venous blood was collected in all patients with aseptic precaution in EDTA anticoagulant for hematological investigation. Separate blood sample was collected for biochemical investigations. The hematological investigations were performed on sysmex KX-21 with standard calibration using fresh whole blood. As a part of CBC, red blood cell indices (MCV, MCH, and MCHC), PCV, RDW, white blood cell count, and platelet count were obtained by sysmex KX-21.

Test methodology for reticulocyte hemoglobin content (CHr)
The test methodology used to measure CHr was flow cytometry. The cellular hemoglobin content of the reticulocytes was measured on a per cell basis by dual angle light scatter and the mean was reported as the CHr.

Specimen collection
Two milliliters of whole blood in a lavender top tube were taken. Specimens were collected and handled carefully, especially when collecting from a central venous catheter.

To ensure accurate results
• Contaminating blood with heparin or saline was avoided.
• Luer adapter to collect specimen directly into lavender top tube was used. Use of syringes was avoided. • Samples were by inverting gently 5 times after collection.
• Specimens were refrigerated promptly while awaiting shipment.
Freezing was avoided.

Interpretation of results
The normal limits for CHr are: 24.5-31.8 pg.
Values <26 pg may be indicative of iron deficiency.

Bone marrow biopsy for evaluation of iron status
To estimate the iron stores directly, usually requires sampling one of the two principal storage depots, the bone marrow or the liver.
Aspiration and biopsy of marrow is usually preferred. More often, the specimen is stained by the Prussian blue method, which renders hemosiderin blue.

Procedure for bone marrow iron estimation
• Under strict aseptic precaution bone marrow aspirates were collected from posterior iliac crest. • Bone marrow smear were freshly collected and contained marrow fragments because iron was demonstrable only in marrow particles. • The smear was fixed in methanol for 15 min and dried.
• Equal volume of 2% potassium ferrocyanide and N/5HCL were mixed in a Coplin jar. The stain was warmed to 55°C just before use. • The stain was then washed in running water for 15 min which was followed by washing in distilled water. • The smear in the slide was counterstained with 1%nuclear fast red or 0.1%neutral red that stained the nuclei red.

Findings
• The smear on the slide was examined and looked for marrow particles with the help of a scanner objective. • The marrow particles were at first examined in low power objective and then using high power objective for presence of iron granules in reticuloendothelial cells and also for free iron.

Criteria For Grading Iron Stains In Bma
Sensitivity and specificity of CHr, SF, SI, TS, and TIBC in males and females in diagnosing IDA were calculated using the following formula. Negative predictive Value= True negative/(True negative+False negative) × 100

Statistical analysis
Data were entered using Microsoft Excel and exported to SPSS version 17.0. Receiver operating characteristic curve analysis was performed to identify the optimal CHr cut off value for predicting IDA. p<0.05 was considered statistically significant.

RESULTS
In the study group of 60 patients, of microcytic hypochromic anemia, 36 (60%) are female and 24 (40%) are male. Ratio of male: female is 1:1.5 (Table 1). Table 2 and Fig. 1 show that patients' age is ranged from 21 to 70 and maximum incidence of 31 (51.66%) patients is seen in age group of 21-30 years. Mean age ±SD of patients in the study group is 36.03±14.21 years.
MCV value of the patients ranging from 40 to 80 fl was found and maximum incidence of 25 (41.66%) patients was seen in range 60-70 fl. Mean±SD of MCV in the study group was 64.74±7.8 fl (Table 5 and Fig. 4).
From Table 13 and Fig. 9, sensitivity and specificity of SI in diagnosing IDA were calculated and found to be 70% and 40%, respectively.
From Table 14 and Fig. 10, sensitivity and specificity of TIBC were calculated and found to be 60% and 40%, respectively.
The sensitivity and specificity of transferrin saturation were calculated and found to be 70% and 40%, respectively (Table 15).       The sensitivity and specificity of transferrin saturation were calculated and found to be 70% and 40%, respectively (Table 15).
The sensitivity and specificity of serum ferritin were calculated and found to be 90% and 80%, respectively (Table 16 and Fig. 11).
The sensitivity and specificity of reticulocyte hemoglobin content (CHr) were calculated and found to be 95% and 80%, respectively (Table 17 and Fig. 12).
The sensitivity and specificity of reticulocyte hemoglobin content (CHr) in males were found to be 91.66% and 75%, respectively (Table 18 and Fig. 13).

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The sensitivity and specificity of reticulocyte hemoglobin content (CHr) in females were calculated and found to be 96.42% and 87.50%, respectively (Table 19 and Fig. 14).

DISCUSSION
The present study had an age distribution of 21-70 years and maximum incidence of 31 (51.66%) patients seen in the age group 21-30 years. Mean±SD of patients in study group is 36.03±14.21 years which correlates with observation by Ahmad et al. [15].        [18].
Maximum patients (n=39, 65%) had transferring saturation in the range of 0.1-10%. The mean±SD of the study group is 13.68±3.22% which is similar to Tater et al. study [18].
Sensitivity and specificity of SI of this study is 70% and 40%, respectively, at a cutoff value off 65 µg/dL compared to studies like Asif et al. [24] where sensitivity and specificity 63.5% and 38.6%, respectively, in diagnosing IDA.
Sensitivity and specificity of serum TIBC of the study is 60% and 40%, respectively, at a cutoff value of 300 µg/dl compared to studies like Asif et al. [24] where sensitivity and specificity 64.5% and 42.8%, respectively, in diagnosing IDA.
Sensitivity and specificity of serum transferrin saturation 70% and 40%, respectively, at a cutoff value of 16% compared to other studies like Fishbane et al. [25] where sensitivity and specificity 81% and 63%, respectively, at a cutoff value off 21%, Tessitone et al. [26] where sensitivity and specificity 59% and 78%, respectively, at a cutoff value of 19%, Kalantar-Zadeh et al. [27] where sensitivity and specificity 88% and 63%, respectively, at a cutoff value of 20% in diagnosing IDA. The variation in sensitivity and specificity is due to different sample sizes.
Sensitivity and specificity of SF 90% and 80%, respectively, at a cutoff value of 10 µg/L compared to other studies like Fishbane et al. [25], where sensitivity and specificity 48% and 75%, respectively, Tessitone et al. [26], where sensitivity and specificity 35% and 78%, respectively, Kalantar-Zadeh et al. [27], 88% and 100%, respectively, in diagnosing IDA. The variation in sensitivity and specificity is due to variations in cutoff values and prevalence of IDA.