Cross sectional study on the pattern of hemotological abnormalities in patients with vitamin B12 deficiency.

Vitamin B12, or cobalamin, is essential for DNA synthesis, red blood cell formation and normal neurological function. Deficiency may arise from inadequate dietary intake, impaired absorption, autoimmune gastritis, gastrointestinal disease or certain medications. Its clinical presentation varies: some patients have no obvious symptoms, while others develop fatigue, pallor, glossitis, paraesthesia, gait disturbance or cognitive changes. The hematological effects range from isolated anemia or macrocytosis to leukopenia, thrombocytopenia and, in severe cases, pancytopenia. The classical picture of megaloblastic anemia may include macro-ovalocytes and hypersegmented neutrophils on peripheral smear, but these findings are not present in every patient. A normal mean corpuscular volume also does not exclude deficiency, particularly when other conditions coexist.

 

Serum vitamin B12 is commonly used to assess vitamin B12 status, although the cutoff for deficiency varies across laboratories and assays. Values below approximately 200-250 pg/mL are generally considered subnormal; borderline results may require interpretation alongside clinical findings and, where available, additional biochemical tests. Complete blood counts and peripheral blood smear examination help identify and classify hematological abnormalities, including changes affecting more than one blood-cell lineage. Recognizing the range of these findings is clinically useful because reliance on macrocytosis alone may fail to identify some affected patients. Systematic assessment of blood counts and smear morphology in patients with vitamin B12 deficiency can describe the frequency and pattern of hematological involvement and help guide further evaluation. This study assessed the hematological abnormalities observed among patients diagnosed with vitamin B12 deficiency at a tertiary-care hospital.

 

AIM

To study the pattern of hematological abnormalities in patients with vitamin B12 deficiency.

 

OBJECTIVES

1.                   To describe the demographic and clinical profile of patients diagnosed with vitamin B12 deficiency.

2.                   To determine the frequency and pattern of anemia, macrocytosis and other complete blood count abnormalities in these patients.

3.                   To describe peripheral blood smear findings and assess their relationship with the hematological profile.

Source of data

Data were obtained from eligible patients diagnosed with vitamin B12 deficiency who attended or were admitted to Seth GS Medical College and KEM Hospital, Mumbai, during the study period. Clinical details and laboratory findings were recorded from patient interviews, examination and hospital laboratory reports.

 

Study design

A hospital-based cross-sectional observational study was conducted.

 

Study location

The study was conducted at Seth GS Medical College and King Edward Memorial (KEM) Hospital, Parel, Mumbai, Maharashtra, India. Patient recruitment and clinical assessment were undertaken through the relevant clinical services, and laboratory investigations were processed by the hospital laboratories.

 

Study duration

The study was conducted over one year.

 

Sample size

The calculated sample size was rounded to 200 participants.

 

Inclusion criteria

·         Patients aged 18 years or older, of either sex.

·         Patients with serum vitamin B12 deficiency based on the hospital laboratory’s validated reference range; a value below 200 pg/mL was used where this was the laboratory cutoff.

·         Patients who provided informed consent to participate.

 

Exclusion criteria

·         Patients who had received vitamin B12 supplementation or treatment before the baseline blood sample was collected.

·         Patients with a known hematological malignancy or a previously diagnosed primary bone marrow disorder.

·         Patients receiving chemotherapy or radiotherapy at the time of assessment.

·         Patients whose essential clinical or laboratory records were unavailable or whose blood sample was inadequate for analysis.

·         Patients who declined consent.

 

Procedure and methodology

Eligible patients were identified consecutively during the study period. After obtaining written informed consent, demographic details, relevant symptoms, medical history, dietary history, medication use and examination findings were recorded using a structured data collection form. Relevant comorbidities and history of gastrointestinal surgery or disorders affecting absorption were also documented.

 

A blood sample was collected for serum vitamin B12 estimation and complete blood count. Hematological variables included hemoglobin, red blood cell count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total leukocyte count, differential leukocyte count and platelet count. Peripheral blood smears were examined for red-cell morphology, macro-ovalocytes, anisopoikilocytosis, hypersegmented neutrophils and other relevant abnormalities. The study findings were classified using the hospital laboratory reference ranges and standard adult hematological criteria. Hematological abnormalities were recorded as present or absent and categorized by cell line and pattern.

 

Sample processing

Blood samples for complete blood count were collected in EDTA tubes, mixed gently and analyzed using the hospital laboratory’s automated hematology analyzer according to the manufacturer’s instructions and the laboratory’s quality-control procedures. Peripheral blood smears were prepared, stained using the laboratory’s routine Romanowsky-type staining method and examined microscopically by trained laboratory personnel. Samples for serum vitamin B12 estimation were collected and processed according to the laboratory’s routine procedure and assay protocol. Results were interpreted using the laboratory’s validated reference range.

 

Statistical methods

Data were entered into a spreadsheet and analyzed using appropriate statistical software. Continuous variables were summarized as mean and standard deviation or median and interquartile range, depending on their distribution. Categorical variables were summarized as frequencies and percentages. The frequency of each hematological abnormality was reported with 95% confidence intervals where appropriate. Associations between categorical variables were assessed using the chi-square test or Fisher’s exact test, as applicable. Continuous variables were compared using the independent-samples t test or Mann-Whitney U test, depending on distribution. A two-sided p value below 0.05 was considered statistically significant.

 

Data collection

Data were recorded on a pretested, structured case record form. The form captured participant identification code, age, sex, clinical features, relevant medical and medication history, serum vitamin B12 result, complete blood count parameters and peripheral smear findings. Each record was checked for completeness before entry, and patient names and other direct identifiers were excluded from the analysis dataset.

Table 1: Pattern of hematological abnormalities by sex

151 of 200 patients (75.5%) had at least one listed hematological abnormality. Anemia was the most frequent finding (64.0%), followed by macrocytosis (41.5%). The sex-based comparisons were not statistically significant for anemia, macrocytosis, leukopenia or thrombocytopenia.

 

Table 2: Demographic and clinical profile by age group

129 participants (64.5%) were younger than 60 years and 71 (35.5%) were aged 60 years or older. Fatigue was reported by 70.0% and paraesthesia by 32.5%.

 

 

 

Table 3: Complete blood count abnormalities by anemia status

Macrocytosis was more frequent among patients with anemia than among those without anemia (57.0% versus 15.3%; p<0.001). Differences in leukopenia, thrombocytopenia and bicytopenia were not statistically significant. Pancytopenia was present in 7 patients (3.5%).

 

Table 4: Peripheral smear findings by macrocytosis status

Hypersegmented neutrophils, macro-ovalocytes, anisopoikilocytosis and a smear pattern suggestive of megaloblastic change were each more frequent in the macrocytosis group (p<0.001 for each comparison).

Table 1 Anemia in 64.0% of participants, macrocytosis in 41.5%, leukopenia in 15.5% and thrombocytopenia in 12.0%. These findings are compatible with the recognized range of blood-count abnormalities in vitamin B12 deficiency, which can include anemia, macrocytosis and one or more cytopenias.

 

The frequency of each abnormality varies across clinical settings and case definitions. Singh et al. (2022) reported anemia in 87.73% and macrocytosis in 88.64% of their 220 cases, while Shilpy et al. (2025) found anemia in 75% but reported that normocytic blood pictures were more common than macrocytic anemia. Differences in referral patterns, deficiency thresholds, coexisting conditions and participant selection may contribute to this variation. In the table, comparisons between male and female participants were not statistically significant for the listed abnormalities.

 

This should be interpreted as a feature of the example data, not as evidence of no sex difference in the study population. Published hospital studies report differing sex distributions and clinical profiles; for example, Singh et al. (2022) reported a slight male majority, while Bandyopadhyay et al. (2021) described deficiency among both male and female symptomatic patients. The estimates should not be directly compared without accounting for differences in study populations and diagnostic criteria.

 

Table 2 Fatigue in 70.0% of participants and paraesthesia in 32.5%. These are clinically plausible symptoms: fatigue may accompany anemia, while paraesthesia may occur with neurological involvement. However, symptom frequencies vary substantially among studies. Agrawal et al. (2024) reported fatigue in 66.7% and tingling or numbness in 54.4% of their patients; Singh et al. (2022) reported paraesthesia in a much higher proportion of their case series. The vegetarian-diet proportion in the table is also not directly comparable with studies that recruited patients based on dietary risk or documented diet in greater detail.

 

Table 3 shows association between anemia and macrocytosis (57.0% versus 15.3%; p<0.001). The association is consistent with the expected relationship between impaired DNA synthesis and enlarged red cells, but macrocytosis is not present in every patient with vitamin B12 deficiency. Studies have described normocytic presentations and variation in red-cell indices, particularly when deficiency is identified before advanced hematological changes or when other causes of anemia coexist. The example table also shows no statistically significant associations between anemia status and leukopenia or thrombocytopenia; those comparisons would need to be recalculated from the actual participant-level data.

 

Table 4 More frequent hypersegmented neutrophils, macro-ovalocytes, anisopoikilocytosis and a megaloblastic-appearing smear in participants classified as macrocytic. These findings fit the conventional blood-film description of megaloblastic change. Nevertheless, a peripheral smear and mean corpuscular volume should be interpreted alongside the clinical picture and biochemical results. Vitamin B12 deficiency can present without the full classical smear pattern, and serum B12 thresholds and confirmatory testing vary. Current NICE guidance recommends interpreting test results in their clinical context and considering further testing when results are indeterminate.

Anemia was the most frequent hematological abnormality, and macrocytosis was associated with anemia. Common smear changes were more frequent in the macrocytosis group. These results are simulated and cannot support conclusions about the actual study population; the conclusion must be rewritten after analysis of the collected data.

 

LIMITATIONS

The values currently shown are rather than observed, so they cannot be used to estimate prevalence, test hypotheses or support a study conclusion. For the proposed cross-sectional study, relevant limitations may include its single-centre, hospital-based setting, which may limit generalizability; its cross-sectional design, which cannot establish temporal or causal relationships; and possible variation in vitamin B12 assay thresholds and laboratory reference ranges. Coexisting iron or folate deficiency, chronic disease and medication use may influence blood counts and smear morphology. If methylmalonic acid or homocysteine testing was not performed, some borderline biochemical results may also be difficult to interpret. These limitations should be retained only where they accurately describe the completed study.

1.       Shipton MJ, Thachil J. Vitamin B12 deficiencya 21st century perspective. Clin Med (Lond). 2015;15(2):145-50. doi:10.7861/clinmedicine.15-2-145.

2.       Mahajan SK, Aundhakar SC. A study of the prevalence of serum vitamin B12 and folic acid deficiency in Western Maharashtra. J Family Med Prim Care. 2015;4(1):64-8. doi:10.4103/2249-4863.152255.

3.       Sen K, Sinhamahapatra P, Lalhmachhuana J, Ray S. A study of clinical profile of vitamin B12 deficiency with special reference to dermatologic manifestations in a tertiary care hospital in Sub-Himalayan Bengal. Indian J Dermatol. 2015;60(4):419. doi:10.4103/0019-5154.160506.

4.       Hannibal L, Lysne V, Bjørke-Monsen AL, Behringer S, Grünert SC, Spiekerkoetter U, et al. Biomarkers and algorithms for the diagnosis of vitamin B12 deficiency. Front Mol Biosci. 2016;3:27. doi:10.3389/fmolb.2016.00027.

5.       Green R. Vitamin B12 deficiency from the perspective of a practicing hematologist. Blood. 2017;129(19):2603-11. doi:10.1182/blood-2016-10-569186.

6.       Langan RC, Goodbred AJ. Vitamin B12 deficiency: recognition and management. Am Fam Physician. 2017;96(6):384-9.

7.       Kaur N, Nair V, Sharma S, Dudeja P, Puri P. A descriptive study of clinico-hematological profile of megaloblastic anemia in a tertiary care hospital. Med J Armed Forces India. 2018;74(4):365-70. doi:10.1016/j.mjafi.2017.11.005.

8.       Kumar GS, Swetha, Rajendran V. A study on incidence of vitamin B12 deficiency in patients with pancytopenia. Int J Adv Med. 2019;6(5):1402-7. doi:10.18203/2349-3933.ijam20194140.

9.       Yajnik CS, Behere RV, Bhat DS, Memane N, Raut D, Ladkat R, et al. A physiological dose of oral vitamin B-12 improves hematological, biochemical-metabolic indices and peripheral nerve function in B-12 deficient Indian adolescent women. PLoS One. 2019;14(10):e0223000. doi:10.1371/journal.pone.0223000.

10.    Wolffenbuttel BHR, Wouters HJCM, Heiner-Fokkema MR, van der Klauw MM. The many faces of cobalamin (vitamin B12) deficiency. Mayo Clin Proc Innov Qual Outcomes. 2019;3(2):200-14. doi:10.1016/j.mayocpiqo.2019.03.002.

11.    Umasanker S, Bhakat R, Mehta S, Rathaur VK, Verma PK, Bhat NK, et al. Vitamin B12 deficiency in children from Northern India: time to reconsider nutritional handicaps. J Family Med Prim Care. 2020;9(9):4985-91. doi:10.4103/jfmpc.jfmpc_712_20.

12.    Bandyopadhyay D, Roy Choudhury J, Mukherjee K. Vitamin B12 deficiency in Eastern India: a hospital-based cross-sectional study. J Clin Diagn Res. 2021;15(4):BC01-BC04. doi:10.7860/JCDR/2021/46642.14685.

13.    Sahu R, Sethy S, Behera M, Parija B. Vitamin B12 deficiency and its correlations with clinical, haematological and electrophysiological parameters: study from a tertiary care hospital of Odisha. Asian J Med Res. 2021;10(4):1-6.

14.    Singh J, Dinkar A, Gupta P, Atam V. Vitamin B12 deficiency in northern India tertiary care: prevalence, risk factors and clinical characteristics. J Family Med Prim Care. 2022;11(6):2381-8. doi:10.4103/jfmpc.jfmpc_650_21.

15.    Silverstein WK, Cheung MC, Lin Y. Vitamin B12 deficiency. CMAJ. 2022;194(24):E843. doi:10.1503/cmaj.220306.

16.    Verma A, Aggarwal S, Garg S, Kaushik S, Chowdhury D. Comparison of serum holotranscobalamin with serum vitamin B12 in population prone to megaloblastic anemia and their correlation with nerve conduction study. Indian J Clin Biochem. 2023;38(1):42-50. doi:10.1007/s12291-022-01027-x.

17.    Wolffenbuttel BHR, Owen PJ, Ward M, Green R. Vitamin B12. BMJ. 2023;383:e071725. doi:10.1136/bmj-2022-071725.

18.    National Institute for Health and Care Excellence. Vitamin B12 deficiency in over 16s: diagnosis and management. NICE guideline NG239. London: NICE; 2024.

19.    Agrawal AR, Mair N, Mehta RS, Chakrapani AS, Gupta K, Srivastav Y, et al. Clinical and hematological characteristics of vitamin B12 deficiency and evaluation of the therapeutic response to vitamin B12 supplementation. Cureus. 2024;16(12):e76468. doi:10.7759/cureus.76468.

20.    Shilpy, Mishra M, Saha SS, Kumar A. Clinical and hematological parameters and their correlation with the severity of vitamin B12 deficiency: a hospital-based study. Cureus. 2025;17(9):e91907. doi:10.7759/cureus.91907.