European Journal of Cardiovascular Medicine

All children aged between 6 months and 5 years admitted in a pre-decided Paediatric unit were screened for Hb levels by Complete Blood Counts (CBC). Total 1552 children were hospitalised during this study period out of which 853 children (54.37%) were between the age group of 6 months to 5 years. 361 children diagnosed out of these 853 had anaemia as per WHO criteria (Hb<11 gm/dl). In all these 361 children serum vitamin B12 levels were performed. For this two ml of serum was collected to estimate serum vitamin B12 levels. Vitamin B12 levels were performed by a competitive chemiluminescent immunoassay.

80 patients out of these 361 had serum vitamin B12 level < 250 pg/dl (as per lower limit defined by Institute of Medicine Standing Committee, USA) and were enrolled after applying exclusion criteria. A detailed history regarding name, age, sex, address, contact number, chief complaints with duration were recorded. Complete birth history, feeding history, dietary history along with antenatal history were recorded. The socioeconomic status of the patients was established based on the Modified Kuppuswamy scale. Complete examination including general and systemic examination and anthropometry was performed.  All the data were then entered into a predesigned proforma for this study. Developmental assessment of these children was performed using Denver II Development Screening Test. Laboratory investigation included complete hemogram, peripheral blood smear and serum vitamin B12 levels.  As there were no previous studies regarding the cut off levels/range for severity of Serum Vitamin B12 deficiency, working criteria to label Mild to moderate and Severe Vitamin B12 deficiency were defined.

Serum vitamin B12 levels between 100-250 pg/dl were considered as mild to moderate vitamin B12 deficiency and less than 100 pg/dl were considered as severe vitamin B12 deficiency.  Cranial magnetic resonance imaging (MRI) was performed in all the patients. All neuroimaging was evaluated by a trained radiologist and findings were noted in the predesigned proforma. Finally all data were entered into Microsoft excel master sheet and presented as frequency, percentage, and/or mean (standard deviation) wherever applicable.

Objective

To find out the Magnetic resonance imaging (MRI) changes in brain with children aged groups 6 month to 5 years having vitamin B12 deficiency with neurological manifestation.

Measurement of Vitamin B12 level

Serum vitamin B12 levels were measured using commercially available Roche E 170 Vitamin B12 ECLIA kit. The Vitamin B12 assay kit employs a competitive test principle using intrinsic factor specific for vitamin B12. Collected blood samples (fasting not compulsory) in appropriate vials were stored in refrigerator and protected from light as per manufacturer’s instructions.

MRI examination

Brain MRI was performed using standard sequences including T1, T2, FLAIR, Diffusion and Gradient echo. For analysis of both the number and volume of White matter hyperintensities (WMH)s, anchored 7-point severity ratings was applied in four regions, including periventricular (i.e., frontal horn, occipital horn, and lateral bands), deep (i.e., frontal, temporal, parietal and occipital lobes), basal ganglia (i.e., caudate nucleus, putamen, Globus pallidus, internal capsule and thalamus), and infratentorial (i.e., mesencephalon, pons, medulla oblongata, and cerebellum) areas.

Statistical analysis

Data collected were entered and compiled in MS Excel spread sheet. Qualitative / categorical variables were presented as frequency, percentage and analysed using Chi square test / Fischer Exact test as applicable. Continuous variables were expressed as mean (standard deviation) and were analysed using t test / Mann Whitney test as applicable.  P value < 0.05 was taken as statistically significant. All statistical analysed were done by appropriate statistical software. 

Out of 80 cases enrolled, 49 (61.25%) were males and 31 (38.75%) were girls. 51(63.75%) cases were between 6 to 15 months. 38(47.50%) cases had severe vitamin B12 deficiency (<100 pg/dl) and remaining 42(52.50%) had mild to moderate vitamin B12 deficiency (100-250 pg/dl). 65%(52/80) cases presented with neurological manifestations and  had significantly lower levels of serum vitamin B12 (90.86±58.78pg/dl) as compared to those not having Neurologic manifestations. Mean vitamin B12 levels were also significantly lower in cases having neuro-development delay, neuro-developmental-regression, seizures, abnormal movements and hypotonia as compared to those who did not have these manifeststions.73.75% (59/80) cases with abnormal MRI brain had significantly lower serum vitamin B12 levels (95.45±56.33pg/dl). Cortical atrophy and white matter changes were predominant findings in 90% cases. Levels of serum vitamin B12 in cases having basal ganglia and cerebellum involvement were lowest (70.50±59.04pg/dl) when compared to only grey matter or white matter involvement (92.05±54.95pg/dl). Bilateral brain atrophy was seen in maximum number of cases. Serum vitamin B12 levels at cut-off of <156pg/dl had sensitivity and specificity of 90% and 62% respectively for abnormal MRI brain.

Table 1 :- Presence or absence of abnormal MRI brain in different categories of serum vitamin B12 deficiency.

Cases with abnormal MRI brain had significantly lower vitamin B12 levels (95.45±56.33 pg/dl) in comparison to cases with normal MRI brain findings (167.42±65.00pg/dl) (P<0.0001).

Table2 :- MRI brain findings in various categories of serum vitamin B12 level deficiency

[A vs B p= 0.816; A vs C p=0.110, A vs D p=0.171, B vs C p=0.165, B vs D p=0.239, C vs D p= 0.914]

Cortical atrophy and white matter changes were predominant findings seen in 90% of cases with abnormal MRI brain. Levels of serum vitamin B12 in cases having basal ganglia and cerebellum involvement were lowest (70.50±59.04 pg/dl) when compared to cases with only grey matter or white matter involvement (92.05±54.95 pg/dl).

Table 3 :- Cortical Changes on MRI brain in various categories of serum vitamin B12 level deficiency

[A vs B p= 0.190; A vs C p=0.535, A vs D p=0.249, A vs E p=0.586, B vs C p=0.423, B vs D p=0.962, B vs E p = 0.368 C vs D p= 0.502, C vs E p = 0.925, D vs E p =0.446, ]

Frontal lobe atrophy was seen in maximum number of cases (90%) having abnormal MRI followed by parietal and temporal lobe atrophy.

Table 4 : White matter changes on MRI brain in various categories of serum vitamin B12 level deficiency

Periventricular changes (delayed myelination) and corpus callosum thinning were seen in maximum number of cases having abnormal MRI brain.

Table 5 :- Basal ganglia changes on MRI brain in various categories of serum vitamin B12 level deficiency

[A vs B p = 0.995, A vs C p=0.909, B vs C p = 0.922]

Caudate lobe and putamen atrophy were predominant findings causing thinning of basal ganglia in cases having abnormal MRI.

Table 6 :- Unilateral/Bilateral MRI brain changes in various categories of serum vitamin B12 level deficiency

Bilateral brain atrophy was seen in maximum number of cases (80%) having abnormal MRI.

Table 7 : Radio-pathologic changes on MRI brain in various categories of serum vitamin B12 level deficiency

[A vs B p = 0.183, A vs C p = 0.927, A vs D p=0.732, B vs C p = 0.303, B vs D p = 0.115, C vs D p = 0.714]

Both simultaneous diffuse restriction and T2 FLAIR hyperintensities were seen in maximum number of cases (78%) having abnormal MRI.

Fig. 1: ROC curve for cut-off serum vitamin B12 level for presence or absence of abnormal MRI finding in cases having serum vitamin B12 deficiency.

Abnormal MRI brain findings in the current study were seen in 73.7% cases. We observed area under the curve of 0.786 (95% C.I. 0.68 – 0.87). With a significant p value (<0.0001) and serum vitamin B12 level at cut-off of ≤ 156 [pg/dl], the sensitivity, specificity, PPV and NPV was 89.83%, 61.90%, 86.90%, and 68.40% respectively for abnormal MRI. Different sensitivity and specificity at different cut-offs of vit B12 have been shown in table 19.

Table 8 : Sensitivity, specificity for abnormal MRI at different levels of serum vitamin B12

In the current study serum vitamin B12 cut-off level of 166.8 pg/dl was found to have sensitivity of 90% and serum vitamin B12 cut-off level of 91.2 pg/dl was found to have a specificity of 90%.

Fig. 2: ROC curve for cut-off MCV level for presence or absence of abnormal MRI finding in cases having serum vitamin B12 deficiency.

Abnormal MRI brain findings in the current study were seen in 73.7% cases. We observed area under the curve of 0.918 (95% C.I. 0.83 – 0.96) with a significant p value (<0.001) and at a cut off value of 92 fl showing sensitivity, specificity, PPV, and NPV as 88.14%, 80.95%, 92.90%, and 70.80% for abnormal MRI. LR ratio +LR=4.63, LR- = 0.15.

Different sensitivity and specificity at different cut-off of MCV has been shown in table 20.

Table 9 : Summary Table for Abnormal MRI findings for MCV

In the current study MCV cut-off level of 88.9 fl was found to have sensitivity of 90% and MCV cut-off level of 96.35fl was found to have a specificity of 90%.

MRI has been considered the exam of choice for detecting B12 deficiency-related central nervous system involvement and for excluding possible mimics. Imaging of the spinal cord in cases of severe myelopathy that are not initially recognized as the result of vitamin B12 deficiency, had characteristic hyperintensity on T2-weighted imaging, described as an inverted V-shaped pattern in the cervical and thoracic spinal cord.

Low levels of vitamin B12 have been associated with neurocognitive disorders. This evidence-based analysis assessed the usefulness of serum vitamin B12 testing as it relates to brain function. Based on low to moderate quality of evidence, treatment with vitamin B12 and folate in patients who have mild cognitive impairment seems to slow the rate of brain atrophy compared with patients who have mild cognitive impairment receiving a placebo. Whether this translates into clinical benefit is unknown. A recent metanalyses conducted by Ford AH, showed that raised total plasma homocysteine is associated with an increased risk of cognitive impairment and dementia, although available evidence from randomized controlled trials shows no obvious cognitive benefit of lowering homocysteine using B12 vitamins.

However, assessing vitamin B12 levels in patients with cognitive impairment or as part of work up of dementia is recommended. Radiologic aspects of low vitamin B12 or Homocysteine include periventricular leukoencephalopathy, manifested as white matter hypodensity on CT scan or hyperintensity on T2 weighted MRI, brain atrophy and silent brain infarcts. Other, neurological disorders also described such as cerebellar ataxia, abnormalities of cranial nerves, Parkinsonian syndrome, mood disturbance (depression and mania) and movement disorders².

In a single case study⁵ 16-month-old infant with vitamin B12 deficient mother and with low socioeconomic level was reported. On admission, the patient was apathic, hypotonic, and lethargic. Serum vitamin B12 level was below detectable limits. On cranial magnetic resonance imaging (MRI), T2-weighted images revealed bilateral frontal and parietal periventricular high-signal symmetric lesions in the white matter (delayed myelination) and frontoparietal cortical atrophy. On day 3 of Vitamin B12 therapy, involuntary movements were observed.

Neuro-radiological manifestation are seen in severe deficiency occurring mostly when serum vitamin B12 levels are <91 pg/dl but subtle/milder neuro-radiological features can also be seen in cases having serum vitamin B12 levels<156 pg/dl. Based on this study it is recommended that serum vitamin B12 levels between 160 to 250 pg/dl, between 90-160 pg/dl and <90 pg/dl should be categorised as mild, moderate and severe vitamin B12 deficiency states respectively. Early diagnosis and treatment is important to prevent neurological morbidity in these patients.

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