Vitamin B₁₂ and vitamin B₉ are essential micronutrients, with shared functions and the deficiency can be associated with public health problems worldwide 1. These two are water soluble vitamins; B₁₂ is found largely in non-vegetarian sources, which is very important for normal neurological function, red cell production, and synthesis of DNA 2. The main sources of vitamin B₁₂(cobalamin) are meat, dairy products, and that of folate (vit B₉) are leafy vegetables, fruits, and legumes as well as dairy products 3.

The human body retains a substantial reserve of vitamin B₁₂, ranging from 2 to 5 milligrams, compared to its daily needs. Consequently, even with severe malabsorption, it typically takes 2 to 5 years for a deficiency of vitamin B₁₂ to manifest 4. The Indian Council of Medical Research recommends daily dietary intakes of vitamin B₁₂ 1μg/day for adults, 1.5 μg/day during pregnancy, and 1.2 μg/day during lactation. A vitamin B₁₂ level of 200 pg/ml or lower is considered as deficient subclinical vitamin B₁₂ insufficiency which is highly prevalent among the elderly and in impoverished countries, vegetarian populations and with excess alcohol.

Unlike vitamin B₁₂, in comparison to daily needs, the body's reserves of folate (5–10 mg) are quite tiny 5. Therefore, folate must be ingested regularly. After many days of dietary folate limitation, serum folate levels fall. Folate deficiency is typically caused by dietary deficiencies and might affect the elderly and similar to people who are hyper-alimentating or haemolysing, population of alcoholics 6. Assessments of folate in serum and red blood cells are used to make the diagnosis. Serum folate levels provide information about the patient's current folate levels, while red cell folate level provides information on folate levels for the six weeks’ prior i.e. lifetime of RBC 7.

The synthetic form of folate is called folic acid. Folate is transformed into (tetra-hydrofolic acid) THF. This substance passes through a number of transfer/methylation processes that are critical for the synthesis of nitrogenous bases in RNA and DNA and are required for the development of red blood cells (RBCs) 8. The inability to produce tetrahydrofolate due to a shortage in either vitamin B₁₂ or folate inhibits cell proliferation in the marrow by causing a malfunction in DNA synthesis, but RNA synthesis and the synthesis of cytoplasmic components are unaffected which further results in megaloblastic anemia⁵ with characteristic hyper-segmented polymorphic nuclear neutrophils 9.

Anaemia is a major problem worldwide and the most frequent hematologic disorder found in newborns and young children. Haemoglobin (Hb), haematocrit, or red blood cell counts that are below the typical age- and sex-adjusted values are considered to be anaemic in clinical practice 10.

Serum and plasma concentrations of these vitamins are the most helpful indicators for determining the status of the population with regard to folate and vitamin B₁₂. Additionally, variations in haematological indices in folate and vitamin B₁₂ deficiency may aid in the diagnosis. These indices include mean corpuscular volume (MCV), red cell distribution width (RDW), mean platelet volume (MPV), platelet distribution width (PDW), RBC, haemoglobin (Hb), and haematocrit (HCT). But there is not much information available regarding these factors 11. The aim of this study was to illustrate the role of these haematological markers in predicting the vitamin B₁₂ and folate status in the body of women thereby preventing the occurrence of anaemia to some extent.

The laboratory reports of total 150 female subjects with vitamin B₁₂ level of age group between 18- 40 years were analysed in this study. Out of this vitamin B₉ was available in 79 cases. Available data related to iron profile, haematological parameters such as haemoglobin and red cell indices were collected.

All data were transferred to Jamovi for analysis. The analysed data was summarised in tables as follows:

The analysis of the dataset reveals a comprehensive array of haematological and nutritional parameters among the participants. Haematological measures include platelet count, white blood cell count, and haemoglobin averages and standard deviations indicative of diverse profiles among the subjects. These findings collectively underscore the complexity of individuals' haematological and nutritional statuses, highlighting the need for further investigation into potential deficiencies and their implications for overall health and well-being. Understanding these parameters provides valuable insights for implementing targeted interventions aimed at optimizing haematological health and mitigating potential health risks within the population (Table 1).

In Table 2, categorised laboratory information is provided as per the normal range.

A Chi-square test was performed to assess the relationship between vitamin B₁₂ and hematological parameters. From Table 3, it is evident that there was no statistically significant association between vitamin B₁₂ and hematological parameters (p-values of p> 0.05 stating their association to be statistically insignificant).

Data from 150 female subjects revealed an average age of 28.19 years (±6.70). Mean vitamin B₁₂ levels were 433.53 pg/ml (±511.19), and mean folate levels were 7.74 ng/ml (±5.49). Substantial variability was observed in iron-related markers and haematological parameters, including haemoglobin, WBC count, and platelet count.

Statistical analysis revealed no significant associations between vitamin B₁₂ and haematological parameters (p > 0.05).

Vitamin B₁₂ or B₉ deficiency is one of the reasons for megaloblastic anaemia which includes hematologic picture such as macrocytic erythrocytes, decreased platelets, decreased leucocytes, increased mean corpuscular volume. In our retrospective study we have studied these hematologic pictures in females by comparing them in two groups normal vitamin B₁₂ level and decreased vitamin B₁₂ level.

In this retrospective study, levels of serum vitamin B₁₂ was available in 150 female subjects between the age group 18 to 40 years during the period Jan 2018 to Dec 2022. Significant percentage of studied population i.e., 42.7% out of 150 were having vitamin B₁₂ deficiency. Serum folate levels were available in 77 subjects, 37.7% out of this had folate deficiency. In an azar cohort study by Azimi et al. 12, 61% of the study population had vitamin B₁₂ deficiency and 16.8% of population had folate deficiency. A study conducted using several Brazilian demographic subgroups found that vitamin B₁₂ deficiency was prevalent in 4.9% of the group under observation, folate deficiency was virtually non-existent (0.3%). Other research have revealed similar findings 13, 14, 15, 16, 17, 18, 19. Low levels of vitamin B₁₂ and folate were found in a significant proportion of participants (98.2% and 27.1%, respectively) in a published study by Fakhrizadeh et al. 20, that examined serum folate and vitamin B₁₂ in healthy Iranian adults. According to Golbahar et al. 21, there is no fortification of grain products with folic acid in their nation, incorrect vegetable preparation, and a high incidence of low folate and vitamin B₁₂ levels in their study. These factors could be the cause of these deficiencies. A study from India conducted by Sivaprasad et al. 22, found that prevalence of B₁₂ deficiency is more among 20 to 60 years aged urban population compared to vitamin B₉ deficiency which is similar to our study. A study conducted by Mishra et al. 23, among children with megaloblastic anaemia found that increased number of children are having vitamin B₁₂ and B₉ deficiency among which girls are more than boys.

In our study there is no association between serum B₁₂ level and haematological markers such as haemoglobin, red cell indices, total leucocyte count. A study conducted by Azimi et al. 12, studied similar association between WBC, MCV, MCH, MCHC, RDW with folate levels found p value >0.05 indicating no statistically significant correlation. While other parameters like RBC, Hb, platelets had p-values <0.05, indicating statistical significance, which goes contrary to our study. In the study, the author also demonstrated no statistically significant association of vitamin B₁₂ values with WBC, MCV, having p-values>0.05 which aligns with our study. While the association of vitamin B₁₂ with Hb and RBC had a p-value <0.05 proving to be statistically significant which is opposite to our study.

The correlation between vitamin B₁₂ concentration and Hb, MCV, MCH, and Hct has been supported by a number of research 24, 25, 26. But most research 27, 28, 29, 30 and the most recent meta-analysis did not establish such a link. The investigation regarding the relationship between haematological markers and the status of vitamin B₁₂ and folate among female patients aged 18-40 years has yielded nuanced insights. Folate and vitamin B₁₂ deficiencies are recognized as global health concerns, with substantial implications for haematological health and overall well-being. While the study revealed a notable prevalence of deficiencies in both vitamins alongside lower-than-normal haemoglobin levels, the direct associations between these deficiencies and haematological parameters appeared complex.

Despite the widespread prevalence of vitamin B₁₂ and folate deficiencies, the study demonstrated limited significant association between haematological parameters and the levels of these vitamins. Notably, haemoglobin levels exhibited no significant associations with either vitamin B₁₂ or folate levels, suggesting that anaemia in this cohort may not be solely attributable to these deficiencies. Similarly, other parameters such as red blood cell count, white blood cell count, and platelet count did not exhibit significant relationships with vitamin B₁₂ or folate levels. However, certain findings hinted at potential connections. For instance, while mean corpuscular volume (MCV) showed a borderline significant association with folate levels, it did not show with vitamin B₁₂ levels. This suggests a nuanced influence of folate status on red blood cell size and haematopoiesis, warranting further investigation.

Despite the study's insightful findings, several limitations need acknowledgment. The retrospective nature of the study and the specific demographic focus on females aged 18-40 years may limit the generalizability of the results. Additionally, demographic details and clinical symptoms and other factors such as dietary habits, genetic predispositions, and comorbidities were not explored but could significantly impact the observed associations. Furthermore, this lack of significant association prompts further investigation into potential underlying reasons. One possibility is that the levels of vitamin B₁₂ and folate within the studied population might not be sufficiently varied to elicit observable effects on haematological parameters. Alternatively, factors such as genetic predispositions, dietary habits, absorption issues, or interactions with other nutrients could influence the relationship between vitamin B₁₂/folate levels and haematological parameters. Furthermore, it is essential to consider the complex interplay between these nutrients and other physiological processes that may indirectly impact haematological parameters. Therefore, additional comprehensive studies with larger and more diverse populations, along with detailed dietary and health assessments, are warranted to better elucidate the intricate relationships between vitamin B₁₂, folate, and haematological parameters. Such investigations are crucial for informing clinical practices and interventions aimed at optimizing haematological health and addressing potential deficiencies in vitamin B₁₂ and folate.

More study is required to determine the impact of other factors, such as homocysteine levels and methyl malonic acid (MMA) on blood folate and vitamin B₁₂ concentrations in order to obtain better and more reliable results.

This study highlights the prevalence of vitamin B₁₂ and folate deficiencies among females aged 18-40 years. Comprehensive screening and management strategies are essential for addressing these deficiencies and optimizing haematological health. Further research is needed to explore interactions between haematological markers and vitamin status, considering dietary, genetic, and health factors.