Anaemia is a condition in which the number of red blood cells (RBCs) and, consequently, their oxygen-carrying capacity is insufficient to meet the body's physiological needs.¹ It affects an estimated 2.36 billion individuals globally, especially women and children.² The World Health Organization defines anaemia as a blood haemoglobin concentration below 13 g/dl in men and below 12 g/dl in women, and further classifies it as mild, moderate or severe according to the degree of haemoglobin reduction.³ In India, national guidelines under the Anemia Mukt Bharat programme have adopted similar age- and sex-specific cut-offs to standardise screening and management across the population.⁴ According to the National Family Health Survey-4 (NFHS-4), 58.4% of children aged 6–59 months, 53.1% of non-pregnant women, 50.3% of pregnant women and 22.7% of men aged 15–49 years were anaemic in India.⁵ The aetiology of anaemia is multifactorial but is, for the most part, preventable, and includes inadequate dietary intake, poor living conditions and a high burden of infections such as malaria and intestinal parasitosis.⁶ Iron deficiency anaemia (IDA), the commonest nutritional cause of anaemia worldwide, is confirmed biochemically by a reduced serum iron and serum ferritin, an increased total iron binding capacity (TIBC) and a reduced transferrin saturation.⁷ Because IDA in adults frequently has an insidious onset, the biochemical diagnosis is often delayed by months or years, allowing time for iron-dependent tissues, including the central nervous system and the retina, to be affected well before the anaemia is recognised clinically. Beyond its haematological role, iron is an essential component of brain growth and myelination, and is required for cell differentiation, protein synthesis, hormone production and cellular energy metabolism.⁸ Iron plays a key role in central nervous system (CNS) functions such as myelination, synaptogenesis and the synthesis of neurotransmitters including dopamine, catecholamines, serotonin and possibly GABA; impulse transmission along the visual pathway might therefore be affected by iron deficiency.⁹⁻¹¹ Oligodendrocytes, which mediate myelination, contain some of the highest concentrations of iron-dependent enzymes among cerebral cells, and a fall in iron availability during a period of active myelination can inhibit oligodendrocyte function and impair the myelination process.¹²,¹³ Iron deficiency has also been associated with dopaminergic dysfunction, and deficiency of retinal dopaminergic function is thought to alter the receptive area of axons and ganglion cells that constitute the retinal nerve fibre layer.¹⁴,¹⁵ Visual evoked potentials (VEP) provide a non-invasive, objective measure of conduction along the optic pathway from the retina to the occipital cortex. The response consists of a sequence of waveforms designated according to their polarity and latency N75, P100 and N145 recorded over the occipital scalp in response to a reversing checkerboard stimulus.¹⁶,¹⁷ Because VEP maturation parallels progressive myelination, and because iron is required for this process, pathway transmission in the visual system may be affected by iron deficiency; delayed myelination and demyelination due to iron deficiency have been shown to increase VEP latency and decrease its amplitude.⁹,¹⁰ Previous studies in infants and growing children have reported prolonged VEP latencies in association with iron deficiency and have proposed VEP as a useful, non-invasive means of detecting subtle nutritional effects on the visual pathway and monitoring the response to iron therapy.¹⁸,¹⁹ Comparable data in adults are more limited and less consistent: some studies have reported prolongation of VEP latency in adult women with IDA, while others have found no significant difference from healthy controls, underscoring the need for further evaluation of VEP in adult iron deficiency anaemia.²⁰,²¹ AIM AND OBJECTIVE To study visual evoked potential in newly diagnosed adult patients of iron deficiency anaemia with haemoglobin less than 10.9 g/dl and compare them with healthy controls.

Study design and setting This was a prospective, case–control study conducted in the Department of Physiology in collaboration with the Department of Medicine and the Regional Institute of Ophthalmology, Pt. B.D. Sharma PGIMS, Rohtak, after approval from the Institutional Ethics Committee. Written informed consent was obtained from all participants in their own language before enrolment. Study population Subjects were divided into two groups: • Group 1 (Cases): 44 newly diagnosed patients of iron deficiency anaemia (IDA) of either sex, aged 18–45 years, with haemoglobin < 10.9 g/dl, confirmed on serum iron studies. • Group 2 (Controls): 44 age- and sex-matched healthy subjects. Exclusion criteria: Chronic disorders (diabetes mellitus, cerebrovascular disease, Parkinsonism, multiple sclerosis, neuromuscular disorders, drug-induced neuropathy, smoking, alcoholism, malabsorption syndromes, chronic hepatic or renal disease); history of intake of drugs with known visual or neurotoxicity; history of loss of vision; altered sensorium or psychiatric illness; pregnancy; glaucoma or any macular pathology; and COVID-19-positive status at the time of testing. Sample size Sample size was calculated using the standard formula for comparison of means between two independent groups [N = (Z₁₋α/2 + Z₁₋β)²(σ1² + σ2²)/(µ1 − µ2)²], with Z₁₋α/2 = 1.96 (95% confidence) and Z₁₋β = 0.84 (80% power), yielding a minimum of 44 subjects per group (total N = 88). Clinical and biochemical assessment Detailed history, general physical examination, complete haemogram and iron profile (serum iron, serum ferritin, TIBC and transferrin saturation) were recorded for every subject on a pre-designed proforma. Visual evoked potential recording Pattern-reversal VEP was recorded on an RMS EMG EP MK2 system using standard silver–silver chloride skin electrodes (impedance < 5 kΩ). The active electrode was placed at Oz (occipital cortex), ground at Fz, and reference electrodes at O1 and O2 (right and left mastoid). The stimulus was a high-contrast (70%) black-and-white checkerboard (8 × 8 min check size, full field ≥ 8°, reversal rate 1.5 Hz, mean luminance 50 cd/m², background luminance 30 cd/m²), viewed monocularly from 100 cm with the fellow eye occluded. Recording parameters were: low filter 2 Hz, high filter 100 Hz, sweep duration 300 ms, 100 epochs averaged, sweep speed 50 ms/division, sensitivity 2 µV/division. Latencies of the N75, P100 and N145 waves and the amplitude of P100 (measured from the preceding N75 trough) were recorded for each eye. Statistical analysis Data were entered in Microsoft Excel and analysed using SPSS version 20. Continuous variables are expressed as mean ± SD and were compared between cases and controls using the unpaired Student's t-test. A p-value < 0.05 was considered statistically significant. Pearson's correlation coefficient (r) was calculated between the study parameters and haemoglobin/iron-profile indices; r < 0.3 was taken as mild, 0.3–0.5 as moderate, and r > 0.5 as strong correlation.

Eighty-eight subjects (44 cases, 44 controls) completed the study. The control group comprised 40 males and 6 females, while the case group comprised 13 males and 31 females (overall male:female ratio 1.45:1). Baseline characteristics and iron profile Table 1. Baseline demographic and anthropometric characteristics of cases and controls (Mean ± SD) Parameter Cases (n=44) Controls (n=44) p-value Age (years) 33 ± 7.4 32 ± 7.5 0.561 Height (m) 1.657 ± 0.186 1.620 ± 0.091 0.239 Weight (kg) 59.568 ± 6.308 60.602 ± 10.282 0.568 BMI (kg/m²) 21.715 ± 2.135 22.966 ± 2.549 0.015* Age, height and weight were comparable between groups; BMI was significantly lower in cases than controls (p = 0.015). Table 2. Comparison of haematological indices and iron profile between cases and controls (Mean ± SD) Parameter Cases (n=44) Controls (n=44) p-value Haemoglobin (g/dl) 7.246 ± 1.468 13.800 ± 1.295 <0.0001* Haematocrit (%) 24.674 ± 4.216 41.838 ± 4.169 <0.0001* MCV (fL) 65.188 ± 9.728 89.125 ± 6.545 <0.0001* MCH (pg) 18.710 ± 3.647 28.387 ± 2.175 <0.0001* MCHC (g/dl) 27.255 ± 3.025 34.516 ± 3.768 <0.0001* Serum iron (µg/dl) 22.685 ± 12.445 100.339 ± 37.989 <0.0001* TIBC (µg/dl) 472.839 ± 65.20 351.662 ± 59.56 <0.0001* Serum ferritin (µg/l) 6.297 ± 3.869 86.502 ± 134.459 <0.0002* Transferrin saturation (%) 6.052 ± 3.383 38.914 ± 11.106 <0.0001* All haematological indices and iron-profile parameters were significantly reduced in cases compared with controls, confirming the biochemical diagnosis of iron deficiency anaemia, except TIBC, which was appropriately elevated in cases. Visual evoked potential findings Table 3. Comparison of VEP latencies (ms) between cases and controls, left eye VEP wave Cases (Mean ± SD) Controls (Mean ± SD) p-value N75 76.186 ± 4.908 73.539 ± 4.987 0.0140* P100 105.131 ± 5.176 98.843 ± 5.422 <0.0001* N145 147.575 ± 5.049 144.056 ± 5.497 0.0024* Table 4. Comparison of VEP latencies (ms) between cases and controls, right eye VEP wave Cases (Mean ± SD) Controls (Mean ± SD) p-value N75 80.261 ± 7.284 73.854 ± 5.994 <0.0001* P100 107.064 ± 5.328 98.161 ± 5.051 <0.0001* N145 146.839 ± 4.924 144.576 ± 6.037 0.0573 VEP latencies of N75 and P100 were significantly prolonged in cases in both eyes. N145 latency was significantly prolonged in the left eye (p = 0.0024) but did not reach significance in the right eye (p = 0.0573). Table 5. Comparison of P100 amplitude (µV) between cases and controls Eye Cases (Mean ± SD) Controls (Mean ± SD) p-value Left eye 3.02 ± 1.734 3.23 ± 1.792 0.5779 Right eye 3.341 ± 1.757 3.57 ± 1.482 0.512 P100 amplitude was lower in cases than controls in both eyes, but the difference did not reach statistical significance. Correlation of VEP with the anaemia/iron profile Table 6. Correlation (Pearson's r) of VEP latencies with haemoglobin and iron profile, left eye Parameter Group N75 P100 N145 Hb Cases 0.275 -0.139 0.134 Control -0.400 -0.160 0.166 S. Iron Cases 0.057 -0.014 0.200 Control 0.112 -0.161 0.025 TIBC Cases -0.247 -0.149 -0.257 Control 0.015 0.039 0.147 S. Ferritin Cases 0.028 0.118 0.252 Control 0.022 0.087 0.290 Trans. Sat. Cases 0.053 0.096 0.490 Control 0.153 -0.010 0.059 Table 7. Correlation (Pearson's r) of VEP latencies with haemoglobin and iron profile, right eye Parameter Group N75 P100 N145 Hb Cases -0.182 -0.043 -0.037 Control 0.501 0.129 0.005 S. Iron Cases 0.031 -0.179 0.034 Control 0.217 0.002 0.220 TIBC Cases -0.037 0.318 0.028 Control -0.134 -0.065 0.063 S. Ferritin Cases 0.153 -0.262 -0.170 Control 0.020 0.056 -0.116 Trans. Sat. Cases 0.080 0.052 -0.059 Control 0.212 -0.099 -0.223 r < 0.3 = mild correlation; 0.3–0.5 = moderate correlation; r > 0.5 = strong correlation. Positive values indicate a positive correlation, negative values a negative correlation. A moderate positive correlation was seen between N75 latency and haemoglobin in controls, and between N145 latency and transferrin saturation, in the left eye. In the right eye, a strong positive correlation (r > 0.5) was observed between haemoglobin and N75 latency in controls, and a moderate positive correlation between TIBC and P100 latency in cases. Correlations for other variables were mild and inconsistent in direction between cases and controls.

The present study demonstrates significant prolongation of VEP latencies in adult patients of IDA compared with age- and sex-matched healthy controls, supporting the hypothesis that iron deficiency causes subclinical electrophysiological impairment of the visual pathway even before overt ocular symptoms develop. Haematological and iron-profile findings All haematological indices and iron-profile parameters were significantly reduced in cases compared with controls, confirming the diagnosis of IDA and consistent with previous reports by Akdogan et al.²² in women, Algarín et al.¹⁹ in infants, and Vinodha et al.²⁰ in adult women, all of whom found significantly lower haemoglobin and iron-profile values in IDA patients than controls. Visual evoked potential findings VEP latencies of N75, P100 and N145 were prolonged in both eyes of IDA patients in this study, with N75 and P100 reaching statistical significance in both eyes and N145 reaching significance in the left eye only; P100 amplitude was reduced but not significantly so. Iron is necessary for normal myelination, and reduced visual and auditory evoked potentials due to hypomyelination have been described in IDA.⁹ These findings are consistent with Sayorwan et al.,²³ who reported significant prolongation of VEP latency related to demyelination, and with Algarín et al.,¹⁹ Monga et al.¹⁸ and Sarici et al.,¹⁶ who reported prolonged VEP latencies in paediatric and infant IDA populations and proposed VEP as a useful non-invasive marker of subclinical visual pathway involvement and of response to iron therapy. Vinodha et al.²⁰ similarly found increased P100 latency in adult women with IDA, though the difference was not statistically significant in their cohort, while Hamzei-Moghaddam et al.²¹ did not find a significant difference in VEP latencies between anaemic and normal female subjects a discrepancy that may reflect differences in severity of anaemia, sample size or age distribution between studies. Wadhera et al.²⁴ similarly documented physiological variation in VEP parameters in relation to age and general health status in a paediatric and adolescent population, lending further support to the role of adequate nutrition, including iron sufficiency, in normal VEP maturation. The present findings, obtained in a mixed-sex adult cohort with confirmed moderate-to-severe IDA, extend this evidence base by demonstrating significant VEP latency prolongation across both eyes. Correlation of VEP with the anaemia profile Correlations between VEP latencies and haemoglobin or iron-profile indices in the present study were mild to moderate and, in several instances, inconsistent in direction between cases and controls or between the two eyes. This pattern has also been observed by other authors and likely reflects the multifactorial determinants of VEP latency including pattern size, contrast, luminance, refractive error and fixation beyond iron status alone. Nonetheless, the overall direction of change, with longer VEP latencies accompanying a more deranged iron profile, is consistent with the underlying biological hypothesis that iron deficiency impairs CNS myelination.

Adult patients with newly diagnosed iron deficiency anaemia (Hb < 10.9 g/dl) show significant prolongation of VEP latencies (N75 and P100 in both eyes, and N145 in the left eye) compared with healthy controls, even though amplitude changes were not statistically significant. These findings indicate subclinical involvement of the visual pathway in adult IDA that precedes overt visual symptoms. VEP is a non-invasive, objective electrophysiological tool that can be used for early detection and monitoring of CNS involvement in patients of iron deficiency anaemia, and may have a role in assessing response to iron replacement therapy. Limitations • The sample size, though adequate as per a priori calculation, was relatively small; a larger cohort would strengthen the reliability of the findings. • This was a case–control study; a longitudinal cohort design would provide stronger evidence of causality. • A prospective pre- and post-treatment design in the same patients would better establish whether VEP changes are reversible with iron supplementation.

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