Abstract
Background Megaloblastic anemia is characterized by abnormally large red blood cells caused by a deficiency in either vitamin B12 or folic acid, both of which are essential for DNA synthesis. Vitamin B12 insufficiency can lead to severe neurological damage, making early identification of vitamin B12 deficiency crucial to prevent irreversible harm. Vitamin B12 deficiency results in decreased levels of holotranscobalamin (Holo-TC) and increased levels of methylmalonic acid (MMA). Methylmalonic acid is considered the gold standard for diagnosing B12 deficiency because it is a specific marker that rises when B12 is insufficient, even when serum B12 levels appear normal. Elevated MMA levels reflect impaired B12 metabolism, making it a critical tool for early detection and intervention. Previous research indicates that Holo-TC, the active form of vitamin B12 available to cells, is a more specific diagnostic tool for early vitamin B12 deficiency than total B12. This study aims to determine the diagnostic validity of total vitamin B12 and Holo-TC using MMA as the gold standard in patients with megaloblastic anemia. Methods A total of 95 megaloblastic anemia patients were selected from Jinnah Hospital and Lahore General Hospital, Lahore, Pakistan, after receiving approval from the ethical review committees. This was a cross-sectional study. Whole blood, serum, and urine samples were collected in ethylenediamine tetraacetic acid (EDTA) vials, gel vials, and urine containers, respectively. The EDTA samples were used for complete blood count measurements using a hematology analyzer (Sysmex-XT 1800i, Sysmex America, Inc., Mundelein, IL), while serum and urine samples were employed for the detection of serum folic acid, cobalamin, Holo-TC, and MMA levels through manual enzyme-linked immunosorbent assay (ELISA) techniques. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of Holo-TC and cobalamin were calculated. Results The majority of patients fell within the age group of 20-70 years, with 57% of them being females and 43% males; Holo-TC exhibited a sensitivity of 98.9% and specificity of 50.00%, with a PPV of 98.90% and NPV of 50%. Vitamin B12 demonstrated a sensitivity of 63% and specificity of 50%, with a PPV of 98.33% and NPV of 2.85%. The diagnostic accuracy of Holo-TC and vitamin B12 was observed to be 97.8% and 63%, respectively. Conclusions Between the two, Holo-TC displays higher diagnostic accuracy than vitamin B12 and can serve as the primary test for patients suspected of having vitamin B12 deficiency.