Introduction: Iron deficiency anemia (IDA) is a major public health issue, particularly in developing countries like India, where malnutrition and limited access to healthcare exacerbate the problem. In the Sub-Himalayan region, the coexistence of IDA with deficiencies in Vitamin B12 and folic acid poses an additional challenge to healthcare providers. This study focuses on the prevalence of these combined deficiencies and aims to shed light on the complex interplay between iron, Vitamin B12, and folic acid metabolism in anemia. Aims: To assess the prevalence of iron deficiency anemia among patients in a tertiary care hospital in the Sub-Himalayan region Materials and Methods: This is a cross-sectional observational study conducted at a tertiary care hospital in the Sub-Himalayan region of India. The study was carried out over a period of 1 year, focusing on patients diagnosed with anemia.100 Patients were included in this study. Result: In our study, 5 (5.0%) patients had Normal Range, 13 (13.0%) patients had Mild Deficiency, 15 (15.0%) patients had Moderate Deficiency and 67 (67.0%) patients had Severe Deficiency. The value of z is 9.1335. The value of p is < .00001. The result is significant at p < .05. In our study, 1 (1.0%) patient had Alfa thalassemia trait, 1 (1.0%) patient had Beta thalassemia trait, 1 (1.0%) patient had HbE carrier, 7 (7.0%) patients had HbE disease, 14 (14.0%) patients had HbE trait and 76 (76.0%) patients had Normal Pattern in HPLC. Conclusion: This study demonstrates a strong correlation between iron deficiency anemia and deficiencies in Vitamin B12 and folic acid in the Sub-Himalayan region of India. The coexistence of these deficiencies complicates the management of anemia and necessitates a more comprehensive diagnostic approach. The findings underline the need for nutritional interventions and public health strategies to address these deficiencies in resource-limited settings.
Vitamin B12 (cobalamin) deficiency has been a known clinical entity for long. Megaloblastic anemia results from abnormal maturation of hematopoietic cells due to faulty DNA synthesis. Two vitamins, cobalamin (vitamin B12) and folic acid are essential for DNA biosynthesis. Deficiency of either of these vitamins results in asynchrony in the maturation of the nucleus and cytoplasm of rapidly regenerating cells. In the hematopoietic system, this asynchrony results in abnormal nuclear maturation with normal cytoplasmic maturation, apoptosis, ineffective erythropoiesis, intramedullary haemolysis, pancytopenia and typical morphological abnormalities in the blood and marrow cells.[1,2] There is evidence that the disease is more common than was previously believed[3] but there are very little Indian data on this[4] especially from the eastern parts including Bengal and the eastern states. A recent Pubmed search with keywords of “vitamin B12 deficiency” or “pernicious anemia” and “India” did not reveal any study from this part of the country. However, there are vitamin B12 deficiency-related studies from other parts of India.[4,5,6]
Among the common causes of anemia in this area, the predominant forms are those having iron deficiency and congenital hemolytic anemia. Three consecutive cases of severe anemia requiring blood transfusions, who had florid cutaneous manifestations in the form of skin hyperpigmentation, were subsequently found to be vitamin B12 deficient and had put to our thoughts that vitamin B12 deficiency could be more common in this region than expected. This prompted us to carry out a study despite our limited set up. We did a prospective study between March 2013 and August 2013 to document the incidence of vitamin B12 deficiency with anemia in our hospital, document the clinical presentation and dietary practices in affected patients.
Cutaneous features of vitamin B12 deficiency are not well described in literature. The first description of hyperpigmentation in vitamin B12 deficiency was given by Dr. Bramwell Cook and this has been documented in subsequent case reports. Some of the described cutaneous manifestations are skin hyperpigmentation, vitiligo, angular stomatitis, and hair changes.[7] Hyperpigmentation of the extremities—especially over the dorsum of the hands and feet, with accentuation over the interphalangeal joints and terminal phalanges—associated with pigmentation of oral mucosa is a characteristic of vitamin B12 deficiency.[8] Interestingly, this hyperpigmentation is more common in dark-skinned patients.[7] The relationship of cutaneous features with degree of vitamin B12 deficiency is, however, not well studied.
Study Design
This is a cross-sectional observational study conducted at a tertiary care hospital in the Sub-Himalayan region of India. The study was carried out over a period of 1 year, focusing on patients diagnosed with anemia.
Study Population
The study population comprised patients presenting with clinical symptoms of anemia, including fatigue, pallor, dizziness, and shortness of breath. The inclusion criteria were as follows:
Inclusion Criteria:
Exclusion Criteria:
Sample Size
A total of 100 patients were included in the study. The sample size was calculated based on the prevalence of anemia in the region, using a confidence interval of 95% and a margin of error of 5%.
Ethical Consideration
The study was conducted after obtaining ethical clearance from the institutional ethics committee. Informed consent was obtained from all participants before enrollment.
Data Collection
Clinical Examination: A detailed clinical history was taken, focusing on dietary habits, socioeconomic status, and symptoms suggestive of anemia.
Blood Sample Collection: Venous blood samples were collected under sterile conditions from all patients.
The following laboratory tests were performed:
Complete Blood Count (CBC): To assess hemoglobin levels, red blood cell indices (MCV, MCH, MCHC), and white blood cell count.
Serum Iron Studies:
Serum Iron
Total Iron Binding Capacity (TIBC)
Serum Ferritin (to confirm iron deficiency)
Vitamin B12 Levels: Measured using an electrochemiluminescence immunoassay.
Serum Folic Acid: Measured using a competitive immunoassay.
Peripheral Blood Smear: To assess morphological features of red blood cells (RBCs), such as microcytic, macrocytic, or hypersegmented neutrophils, which are suggestive of nutritional deficiencies.
Statistical Analysis
For statistical analysis, data were initially entered into a Microsoft Excel spreadsheet and then analyzed using SPSS (version 27.0; SPSS Inc., Chicago, IL, USA) and GraphPad Prism (version 5). Numerical variables were summarized using means and standard deviations, while categorical variables were described with counts and percentages. Two-sample t-tests, which compare the means of independent or unpaired samples, were used to assess differences between groups. Paired t-tests, which account for the correlation between paired observations, offer greater power than unpaired tests. Chi-square tests (χ² tests) were employed to evaluate hypotheses where the sampling distribution of the test statistic follows a chi-squared distribution under the null hypothesis; Pearson's chi-squared test is often referred to simply as the chi-squared test. For comparisons of unpaired proportions, either the chi-square test or Fisher’s exact test was used, depending on the context. To perform t-tests, the relevant formulae for test statistics, which either exactly follow or closely approximate a t-distribution under the null hypothesis, were applied, with specific degrees of freedom indicated for each test. P-values were determined from Student's t-distribution tables. A p-value ≤ 0.05 was considered statistically significant, leading to the rejection of the null hypothesis in favour of the alternative hypothesis.
Correlation Analysis: The strength and direction of the relationship between IDA and Vitamin B12/folic acid deficiency were evaluated.
Regression Analysis: Multivariable regression was used to control for confounding factors like age, gender, and dietary habits.
Outcome Measures
The primary outcome of the study was to determine the prevalence of iron deficiency anemia in conjunction with Vitamin B12 and folic acid deficiencies. Secondary outcomes included the severity of anemia, the clinical profile of patients with combined deficiencies, and the effectiveness of combined therapy in patients with multiple deficiencies.
|
Frequency |
Percent |
|
Serum Iron Level-microg/dl Group |
Normal Range |
5 |
5.0% |
Mild Deficiency |
13 |
13.0% |
|
Moderate Deficiency |
15 |
15.0% |
|
Severe Deficiency |
67 |
67.0% |
|
Total |
100 |
100.0% |
|
HPLC |
Alfa thalassemia trait |
1 |
1.0% |
Beta thalassemia trait |
1 |
1.0% |
|
HbE carrier |
1 |
1.0% |
|
HbE disease |
7 |
7.0% |
|
HbE trait |
14 |
14.0% |
|
Normal Pattern |
76 |
76.0% |
|
Total |
100 |
100.0% |
Table 2: Distribution of mean Age
|
Number |
Mean |
SD |
Minimum |
Maximum |
Median |
Age |
100 |
49.1300 |
19.3473 |
13.0000 |
90.0000 |
50.0000 |
|
Number |
Mean |
SD |
Minimum |
Maximum |
Median |
Haemoglobin level at presentaion gm/dl |
100 |
7.3480 |
2.1920 |
2.8000 |
13.1000 |
7.1000 |
Serum Iron Level-microg/dl |
100 |
35.2273 |
32.9697 |
12.2300 |
230.0000 |
23.8750 |
Serum Ferritin Level- ng/ml |
100 |
142.4011 |
223.5200 |
3.0000 |
1000.0000 |
18.3000 |
TIBC-microgram/dl |
100 |
335.3868 |
114.8410 |
103.1200 |
550.0000 |
338.0000 |
Trasferrin Saturation% |
100 |
12.8998 |
13.7117 |
2.8800 |
86.3900 |
8.3950 |
Folic Acid Level-ng/ml |
100 |
10.5826 |
6.2681 |
1.0000 |
23.9700 |
9.8850 |
Correlations |
|
||
VITAMIN B12 LEVEL-pg/ml |
Remarks |
||
Haemoglobin level at presentaion gm/dl |
Pearson Correlation Coefficient (r) |
-0.187 |
Negative correlation |
p-value |
0.062 |
Not Significant |
|
Number |
100 |
|
|
Serum Iron Level-microg/dl |
Pearson Correlation Coefficient (r) |
-0.138 |
Negative correlation |
p-value |
0.172 |
Not Significant |
|
Number |
100 |
|
|
Serum Ferritin Level- ng/ml |
Pearson Correlation Coefficient (r) |
.221* |
Positive correlation |
p-value |
0.027 |
Significant |
|
Number |
100 |
|
|
TIBC-microgram/dl |
Pearson Correlation Coefficient (r) |
-0.102 |
Negative correlation |
p-value |
0.315 |
Not Significant |
|
Number |
100 |
|
|
Trasferrin Saturation% |
Pearson Correlation Coefficient (r) |
-0.124 |
Negative correlation |
p-value |
0.22 |
Not Significant |
|
Number |
100 |
|
|
Folic Acid Level-ng/ml |
Pearson Correlation Coefficient (r) |
.409** |
Positive correlation |
p-value |
<.0001 |
Significant |
|
Number |
100 |
|
Correlations |
|||
FOLIC ACID LEVEL-ng/ml |
Remarks |
||
Haemoglobin level at presentaion gm/dl |
Pearson Correlation Coefficient (r) |
-0.049 |
Negative correlation |
p-value |
0.625 |
Not Significant |
|
Number |
100 |
||
Serum Iron Level-microg/dl |
Pearson Correlation Coefficient (r) |
0.11 |
Positive correlation |
p-value |
0.274 |
Not Significant |
|
Number |
100 |
||
Serum Ferritin Level- ng/ml |
Pearson Correlation Coefficient (r) |
0.081 |
Positive correlation |
p-value |
0.425 |
Not Significant |
|
Number |
100 |
||
TIBC-microgram/dl |
Pearson Correlation Coefficient (r) |
0.147 |
Positive correlation |
p-value |
0.143 |
Not Significant |
|
Number |
100 |
||
Trasferrin Saturation% |
Pearson Correlation Coefficient (r) |
-0.033 |
Negative correlation |
p-value |
0.743 |
Not Significant |
|
Number |
100 |
||
Vitamin B12 Level-pg/ml |
Pearson Correlation Coefficient (r) |
.409** |
Positive correlation |
p-value |
<.0001 |
Significant |
|
Number |
100 |
In our study, 5 (5.0%) patients had Normal Range, 13 (13.0%) patients had Mild Deficiency, 15 (15.0%) patients had Moderate Deficiency and 67 (67.0%) patients had Severe Deficiency. The value of z is 9.1335. The value of p is < .00001. The result is significant at p < .05. In our study, 1 (1.0%) patient had Alfa thalassemia trait, 1 (1.0%) patient had Beta thalassemia trait, 1 (1.0%) patient had HbE carrier, 7 (7.0%) patients had HbE disease, 14 (14.0%) patients had HbE trait and 76 (76.0%) patients had Normal Pattern in HPLC. The value of z is 10.8988. The value of p is < .00001. The result is significant at p < .05. In above table showed that the mean Age (mean±s.d.) of patients was 49.1300± 19.3473. In above table showed that the mean Haemoglobin level at presentaion gm/dl (mean±s.d.) of patients was 7.3480± 2.1920. In above table showed that the mean Serum Iron Level-microg/dl (mean±s.d.) of patients was 35.2273± 32.9697. In above table showed that the mean Serum Ferritin Level- ng/ml (mean±s.d.) of patients was 142.4011± 223.5200. In above table showed that the mean TIBC-microgram/dl (mean±s.d.) of patients was 335.3868± 114.8410. In above table showed that the mean Trasferrin Saturation% (mean±s.d.) of patients was 12.8998± 13.7117. In above table showed that the mean Folic Acid Level-ng/ml (mean±s.d.) of patients was 10.5826± 6.2681.
Haemoglobin level at presentaion gm/dl
The value of Pearson Correlation Coefficient (r) was -0.187. The Negative correlation was found between VITAMIN B12 LEVEL-pg/ml vs Haemoglobin level at presentaion gm/dl. The P-Value was 0.062. The result was not statistically significant.
Serum Iron Level-microg/dl
The value of Pearson Correlation Coefficient (r) was -0.138. The Negative correlation was found between VITAMIN B12 LEVEL-pg/ml vs Serum Iron Level-microg/dl. The P-Value was 0.172. The result was not statistically significant.
Serum Ferritin Level- ng/ml
The value of Pearson Correlation Coefficient (r) was .221*. The Positive correlation was found between VITAMIN B12 LEVEL-pg/ml vs Serum Ferritin Level- ng/ml. The P-Value was 0.027. The result was statistically significant.
TIBC-microgram/dl
The value of Pearson Correlation Coefficient (r) was .221*. The Positive correlation was found between VITAMIN B12 LEVEL-pg/ml vs TIBC-microgram/dl. The P-Value was 0.315. The result was Not Significant.
Trasferrin Saturation%
The value of Pearson Correlation Coefficient (r) was -0.124. The Negative correlation was found between VITAMIN B12 LEVEL-pg/ml vs Trasferrin Saturation%. The P-Value was 0.22. The result was Not Significant.
Folic Acid Level-ng/ml
The value of Pearson Correlation Coefficient (r) was .409**. The Positive correlation was found between VITAMIN B12 LEVEL-pg/ml vs Folic Acid Level-ng/ml. The P-Value was <.0001. The result was Significant.
Folic Acid Level-ng/ml
The value of Pearson Correlation Coefficient (r) was .409**. The Positive correlation was found between VITAMIN B12 LEVEL-pg/ml vs Folic Acid Level-ng/ml. The P-Value was <.0001. The result was Significant.
Haemoglobin level at presentaion gm/dl
The value of Pearson Correlation Coefficient (r) was -0.049. The Negative correlation was found between VITAMIN B12 LEVEL-pg/ml vs Haemoglobin level at presentaion gm/dl. The P-Value was 0.625. The result was not Significant.
Serum Iron Level-microg/dl
The value of Pearson Correlation Coefficient (r) was 0.11. The Positive correlation was found between VITAMIN B12 LEVEL-pg/ml vs Serum Iron Level-microg/dl. The P-Value was 0.274. The result was not Significant.
Serum Ferritin Level- ng/ml
The value of Pearson Correlation Coefficient (r) was 0.081. The Positive correlation was found between VITAMIN B12 LEVEL-pg/ml vs Serum Ferritin Level- ng/ml. The P-Value was 0.425. The result was not Significant.
TIBC-microgram/dl
The value of Pearson Correlation Coefficient (r) was 0.147. The Positive correlation was found between VITAMIN B12 LEVEL-pg/ml vs TIBC-microgram/dl. The P-Value was 0.143. The result was not Significant.
Trasferrin Saturation%
The value of Pearson Correlation Coefficient (r) was -0.033. The Negative correlation was found between VITAMIN B12 LEVEL-pg/ml vs Trasferrin Saturation%. The P-Value was 0.743. The result was not Significant.
Vitamin B12 Level-pg/ml
The value of Pearson Correlation Coefficient (r) was .409**. The Positive correlation was found between VITAMIN B12 LEVEL-pg/ml vs Vitamin B12 Level-pg/ml. The P-Value was <.0001. The result was Significant.
In our study, out of 100 patients most of the patients were 61-70 years old [21(21.0%)]. It was statistically significant (p=.00028), (z=3.6348)
Chithambaram NS et al [9] (2016) examined that all children aged between 6 months to 13 years, admitted in paediatric ward of this hospital for any complaints, were evaluated for anaemia. All patients with Hemoglobin levels less than the WHO cut off levels for anaemia were included in the study.
We found that, male [59(59%)] population was higher than the female population [41 (41.0 %)]. It was statistically significant (p=.01078), (z=2.5456).
Bhardwaj A et al [10] (2013) showed that Under-nutrition (BMI < 18.5 kg/m2) was observed among 68.9% of adolescents (male: 67.1%; female: 70.7; P = 0.29). Anemia was observed to be prevalent among 87.2% males and 96.7% females (P = 0.00).
We found that, significantly higher of patients had Severe Deficiency Serum Iron Level-microg/dl [67(67.0%)]. It was statistically significant (p< .00001), (z=9.1335)
We examined that, most of the patients had HbE trait [14(14.0%)]. It was statistically significant (p< .00001), (z=10.8988)
In our study, the mean Age of patients was [49.1300±19.3473], the mean Haemoglobin level at presentaion gm/dl of patients was [7.3480±2.1920], Zeeshan F et al [11 ](2017) observed that the mean Hemoglobin (Hb) of children was 8 g/dl. Only 4% children had low ferritin level while 60% had low folic acid and 45% had decreased VitB12. There was significant correlation between Hb of mother and child (p =0.02), Vit B12 deficiency (p=0.008) and iron deficiency (p<0.001).
the mean Serum Iron Level-microg/dl of patients was [35.2273 ±32.9697], Yildirim T et al [12 ](2015) showed that Iron, vitamin B12, and folic acid deficiencies were evaluated. Iron deficiency anemia was diagnosed when anemia with iron level <60 μg/dl and ferritin level <12 ng/ml. Vitamin B12 deficiency anemia was diagnosed when anemia with vitamin B12 level <200 pg/ml.
the mean Serum Ferritin Level- ng/ml of patients was [142.4011±223.5200], the mean TIBC-microgram/dl of patients was [335.3868±114.8410], the mean Trasferrin Saturation % of patients was [12.8998±13.7117], the mean Vitamin B12 LEVEL-pg/ml of patients was [376.2683±369.0408], Yildirim T et al [12] (2015) showed that Vitamin B12 deficiency anemia was diagnosed when anemia with vitamin B12 level <200 pg/ml. Folic acid deficiency anemia was diagnosed when anemia with folic acid level <2.6 ng/ml. Prevalence of anemia was found 7.3%. Prevalence of iron deficiency, vitamin B12, and folic acid deficiency were found 7.1%, 64.2% and 10.9%, and 10.9%, respectively the mean Folic Acid Level-ng/ml of patients was [10.5826±6.2681].
This study demonstrates a strong correlation between iron deficiency anemia and deficiencies in Vitamin B12 and folic acid in the Sub-Himalayan region of India. The coexistence of these deficiencies complicates the management of anemia and necessitates a more comprehensive diagnostic approach. The findings underline the need for nutritional interventions and public health strategies to address these deficiencies in resource-limited settings.