Iron deficiency anemia (IDA) is one of the most prevalent hematological disorders in the world and is a significant cause of fatigue, decreased physical function, decreased productivity and poor quality of life. While IDA is frequently viewed as a straightforward nutrition disorder, the clinical presentation is influenced by dietary factors, chronic blood loss, gastrointestinal disease, menstrual losses, pregnancy, inflammation and social factors that delay diagnosis. Recent reviews have stressed that anemia is just one symptom of iron depletion and that biochemical iron deficiency can lead to symptoms before hemoglobin drops below the traditional limits [1],[2]. In patients with a suspected anemia the first investigation is typically a complete blood count (CBC). Classical IDA results in low hemoglobin, low mean corpuscular volume (MCV), low mean corpuscular hemoglobin (MCH), elevated red cell distribution width (RDW), low serum ferritin and reduced transferrin saturation. Interpretation may be challenging, however, when inflammation causes an increase in ferritin or when thalassemia trait and anemia of chronic disease or mixed deficiencies occur simultaneously. Guidelines thus suggest to include CBC indices in addition to iron studies and clinical context and not just hemoglobin [3],[4]. A number of parameters of the red cells and reticulocytes have been investigated to differentiate iron-restricted erythropoiesis from other microcytic conditions. RDW is a measure of anisocytosis and may be elevated early in IDA, and the hemoglobin content of the reticulocytes may be a reflection of the availability of iron to the marrow in the recent past. In iron deficiency, platelet count also increases, and this sometimes is so severe that it is a concern for myeloproliferative disease. These are easy to obtain indices that are especially valuable in resource-limited areas where ferritin and transferrin saturation might not be readily available [5],[6]. Although the literature is voluminous, many clinical laboratories continue to report CBC data without any formal patterns to interpret the severity. The aim of the present study was to evaluate the hematological alterations in adults suffering from IDA and to correlate CBC parameters with biochemical iron status and severity of anemia. The goal was to find practical hematological indicators which could be useful for early suspicion, IDA stratification and monitoring in the routine outpatient setting [7].

The study was conducted as an analytical cross sectional study in the department of Pathology and General Medicine of tertiary care hospital over a period of 12 months. Adult patients (18-65 years) who had symptoms suggestive of anemia and laboratory evidence of iron deficiency were recruited consecutively. A control group of non-anemic adults who attended the health-check services during the same period were included for comparison.Iron deficiency anemia was diagnosed when hemoglobin levels were < 13 g/dL in men and < 12 g/dL in women, serum ferritin was < 30 ng/mL, and/or microcytic/hypochromic indices were present with compatible levels. Patients with chronic kidney disease, known malignancy, acute infection, hemoglobinopathy, recent blood transfusion, pregnancy, active bleeding requiring emergency care or current hematinic treatment were excluded. A total of 120 IDA patients and 60 controls were included in the final sample. Demographic information, dietary pattern, menstrual history, gastrointestinal symptoms and medication history were obtained after written informed consent. Venous blood was drawn in EDTA and plain tubes. CBC was done by a 5 part automated hematology analyzer with daily internal quality control. Serum ferritin, serum iron, total iron-binding capacity and C-reactive protein levels were determined by standard laboratory techniques. Peripheral smears were examined for red cell morphology and platelet estimate. Severity of anemia was classified as mild, moderate or severe based on hemoglobin levels. The data were analyzed using SPSS version 26. All continuous variables were presented as mean ± SD and categorical variables were presented as numbers and percentages. Independent t-test, one-way ANOVA with Tukey post hoc test, chi-square test and Pearson correlation were used. A p-value of < 0.05 was deemed statistically significant.

The study included 180 participants: 120 patients with IDA and 60 controls. The mean age of IDA patients was 35.8 ± 11.2 years, and 78 patients (65.0%) were women. Among cases, 32 (26.7%) had mild anemia, 61 (50.8%) had moderate anemia and 27 (22.5%) had severe anemia. Menstrual blood loss was the most common clinical association among women, while chronic dyspeptic symptoms and low dietary iron intake were frequent among men. Table 1. Baseline hematological and biochemical parameters in IDA patients and controls. Parameter IDA patients (n=120) Controls (n=60) p-value Age (years) 35.8 ± 11.2 34.6 ± 10.4 0.486 Hemoglobin (g/dL) 8.9 ± 1.4 13.2 ± 1.0 <0.001 MCV (fL) 68.7 ± 8.1 86.5 ± 5.7 <0.001 MCH (pg) 20.9 ± 3.8 29.1 ± 2.1 <0.001 RDW (%) 18.9 ± 2.6 13.2 ± 1.1 <0.001 Platelet count (×10^3/µL) 412 ± 124 270 ± 73 <0.001 Serum ferritin (ng/mL) 10.8 ± 6.4 62.3 ± 28.5 <0.001 Transferrin saturation (%) 8.7 ± 4.2 26.9 ± 8.6 <0.001 Table 2. CBC changes according to severity of iron deficiency anemia. Parameter Mild (n=32) Moderate (n=61) Severe (n=27) p-value Hemoglobin (g/dL) 10.8 ± 0.5 8.9 ± 0.6 6.8 ± 0.7 <0.001 MCV (fL) 74.9 ± 6.3 68.5 ± 6.7 61.9 ± 7.4 <0.001 MCH (pg) 23.8 ± 2.7 20.6 ± 2.9 17.8 ± 3.2 <0.001 RDW (%) 16.8 ± 1.9 19.1 ± 2.2 21.0 ± 2.5 <0.001 Platelet count (×10^3/µL) 346 ± 92 423 ± 111 478 ± 139 <0.001 Ferritin (ng/mL) 16.4 ± 6.8 9.9 ± 4.8 5.7 ± 3.1 <0.001 Table 3. Correlation of iron markers with hematological indices in IDA patients. Variable pair Correlation coefficient (r) p-value Interpretation Ferritin vs MCV 0.58 <0.001 Moderate positive Ferritin vs RDW -0.49 <0.001 Moderate inverse Hemoglobin vs platelet count -0.46 <0.001 Moderate inverse Transferrin saturation vs MCH 0.55 <0.001 Moderate positive CRP vs ferritin 0.21 0.023 Weak positive RDW vs severity grade 0.62 <0.001 Strong positive Peripheral smear examination showed microcytic hypochromic red cells in 110 cases (91.7%), anisopoikilocytosis in 101 cases (84.2%), pencil cells in 43 cases (35.8%) and target cells in 18 cases (15.0%). Reactive thrombocytosis above 450 ×10^3/µL was found in 38 cases (31.7%), with no abnormal platelet morphology suggesting a primary platelet disorder. On multivariable analysis, RDW above 16.5%, MCV below 76 fL and ferritin below 15 ng/mL independently predicted moderate-to-severe IDA. The combination of MCV and RDW correctly classified severity in 82.5% of cases, while the addition of platelet count improved classification to 86.7%.

The present study shows that IDA results in a consistent hematological profile, including low hemoglobin, microcytosis, hypochromia, high RDW, low ferritin and low transferrin saturation. These results are in line with the pathophysiology of iron-restricted hemoglobin synthesis reported in the major reviews and support the continued use of CBC indices in situations where biochemical testing might be delayed [8,9]. The RDW was gradually raised as severity progressed and was inversely related to ferritin. This confirms the idea that anisocytosis is a reflection of a changing population of older normocytes and younger iron-restricted microcytes. Previous studies on the classification of anemia have demonstrated that RDW can enhance the interpretation of microcytosis and aid in the differentiation of IDA from some hereditary microcytic anemias in combination with MCV and clinical information [10],[11]. Another significant finding was the negative correlation of hemoglobin with platelet counts. Reactive thrombocytosis is a known but under-reported hematological alteration that occurs in IDA. The mechanism is not completely understood, but, marrow stimulation and changes in thrombopoietic signaling and shared progenitor responses may play a role. Acknowledgment of this association can help avoid unnecessary evaluation for primary thrombocytosis when there is no other iron deficiency anemia present [12],[13]. Depleted iron stores were best identified by serum ferritin, although there was a weak positive correlation between CRP and ferritin in our data, indicating a practical limitation. Deficiency can be masked by inflammatory states, which increase ferritin; in such cases, transferrin saturation, soluble transferrin receptor or reticulocyte hemoglobin may be useful [14],[15]. The study has practical implications in primary care and district level laboratories. Low MCV, high RDW and disproportionately elevated platelets should raise suspicion for iron studies and blood loss, diet, malabsorption or chronic inflammation. Early recognition is crucial as untreated IDA can have a negative effect on cognition, exercise tolerance and maternal health, and can delay diagnosis of gastrointestinal disease in adults [16]. Some limitations are that the study was conducted in a single center, pregnant women were excluded, and there was no follow-up after iron therapy. However, the biochemical confirmation and smear review adds to the validity of observed patterns. Further research is needed to test the ability of automated flagging algorithms based on MCV, RDW, platelet count and ferritin to enhance early detection in high-volume laboratories [17]. A small group of patients with elevated C-reactive protein (CRP) showed a difference between red cell indices and biochemical markers, another clinically relevant finding. The cases demonstrate the need to consider ferritin in conjunction with other inflammatory data in adults. The patient may have low functional iron availability and the ferritin may be borderline, as it is an acute phase reactant. In these situations, a combination of interpretation of transferrin saturation, RDW, smear morphology and treatment response will avoid underdiagnosis. There are also implications for follow-up, depending on the severity-wise pattern. If the iron deficiency is uncomplicated, reticulocyte response will occur within 1-2 weeks of proper iron therapy, and then hemoglobin will slowly increase. When MCV, RDW and platelet count do not improve with adherence, clinicians should consider continuing to evaluate for blood loss, malabsorption, mixed nutritional deficiency or another diagnosis. Therefore, CBC parameters not only serve as a diagnostic tool, but also as a tool to monitor the biological recovery [17]. The results could also help to prioritize patients for further evaluation. In postmenopausal women and adult men with confirmed IDA, care must be taken to assess for occult gastrointestinal bleeding and in reproductive-age women to assess for menstrual blood loss and dietary and pregnancy-related needs. A hematology report which includes a comment on the pattern of microcytosis, anisocytosis and thrombocytosis may therefore be more useful in making a clinical decision than isolated numbers [17].

Significant reductions in hemoglobin, MCV, MCH, ferritin and transferrin saturation and increased RDW and platelet count were associated with iron deficiency anemia. There was a meaningful relationship between RDW, MCV and platelet count with the severity of anemia and these parameters can be used to help suspect and stratify anemia in routine practice. CBC interpretation with iron studies is a practical and useful way to make a prompt diagnosis and proper evaluation of underlying causes

1. Camaschella C. Iron-deficiency anemia. N Engl J Med. 2015;372(19):1832-1843. doi:10.1056/NEJMra1401038. PMID:25946282. 2. Lopez A, Cacoub P, Macdougall IC, Peyrin-Biroulet L. Iron deficiency anaemia. Lancet. 2016;387(10021):907-916. doi:10.1016/S0140-6736(15)60865-0. PMID:26314490. 3. Snook J, Bhala N, Beales ILP, et al. British Society of Gastroenterology guidelines for the management of iron deficiency anaemia in adults. Gut. 2021;70(11):2030-2051. doi:10.1136/gutjnl-2021-325210. PMID:34497146. 4. Pasricha SR, Tye-Din J, Muckenthaler MU, Swinkels DW. Iron deficiency. Lancet. 2021;397(10270):233-248. doi:10.1016/S0140-6736(20)32594-0. PMID:33285139. 5. Auerbach M, Adamson JW. How we diagnose and treat iron deficiency anemia. Am J Hematol. 2016;91(1):31-38. doi:10.1002/ajh.24201. PMID:26408108. 6. Powers JM, Buchanan GR. Diagnosis and management of iron deficiency anemia. Hematol Oncol Clin North Am. 2014;28(4):729-745. doi:10.1016/j.hoc.2014.04.007. PMID:25064710. 7. Guyatt GH, Oxman AD, Ali M, Willan A, McIlroy W, Patterson C. Laboratory diagnosis of iron-deficiency anemia: an overview. J Gen Intern Med. 1992;7(2):145-153. doi:10.1007/BF02598003. PMID:1487761. 8. Cappellini MD, Musallam KM, Taher AT. Iron deficiency anaemia revisited. J Intern Med. 2020;287(2):153-170. doi:10.1111/joim.13004. PMID:31665543. 9. Brugnara C. Iron deficiency and erythropoiesis: new diagnostic approaches. Clin Chem. 2003;49(10):1573-1578. doi:10.1373/49.10.1573. PMID:14500582. 10. Bessman JD, Gilmer PR Jr, Gardner FH. Improved classification of anemias by MCV and RDW. Am J Clin Pathol. 1983;80(3):322-326. doi:10.1093/ajcp/80.3.322. 11. Urrechaga E, Borque L, Escanero JF. Erythrocyte and reticulocyte parameters in iron deficiency and thalassemia. J Clin Lab Anal. 2011;25(3):223-228. doi:10.1002/jcla.20462. 12. Kadikoylu G, Yavasoglu I, Bolaman Z, Senturk T. Platelet parameters in women with iron deficiency anemia. J Natl Med Assoc. 2006;98(3):398-402. PMID:16573305. 13. Demir C, Demir H, Esen R, Atmaca M, Efe S. Altered platelet parameters in iron deficiency anemia. Platelets. 2014;25(1):61-65. doi:10.3109/09537104.2012.758352. PMID:23441975. 14. Cook JD. Diagnosis and management of iron-deficiency anaemia. Best Pract Res Clin Haematol. 2005;18(2):319-332. doi:10.1016/j.beha.2004.08.022. PMID:15737893. 15. Punnonen K, Irjala K, Rajamaki A. Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron deficiency. Blood. 1997;89(3):1052-1057. PMID:9028330. 16. Johnson-Wimbley TD, Graham DY. Diagnosis and management of iron deficiency anemia in the 21st century. Therap Adv Gastroenterol. 2011;4(3):177-184. doi:10.1177/1756283X11398736. PMID:21694802. 17. Powers JM, O'Brien SH. New diagnostic and treatment approaches to iron deficiency. Hematology Am Soc Hematol Educ Program. 2019;2019(1):446-452. doi:10.1182/hematology.2019000039. PMID:31808875.