Keywords
megaloblastic anemia - tropical sprue - vitamin B
12
- folate - pancytopenia - intraepithelial lymphocytosis
Introduction
Megaloblastic anemia is characterized by the distinctive morphological appearance
of developing hematopoietic cells in the bone marrow, resulting from nuclear cytoplasmic
asynchrony, occurring as a result of defective deoxyribonucleic acid (DNA) synthesis.[1] The cause is usually nutritional deficiency or malabsorption of either cobalamin
(vitamin B12) and/or folate. Malabsorption can result from gastrectomy, tropical and nontropical
sprue, intestinal resection, and drugs. It can also occur in defects of metabolism
of these vitamins.[2] While pernicious anemia is reported to be the common cause for megaloblastic anemia
in Western countries, dietary deficiency and malabsorption are the common causes in
India.[3]
[4] Duodenal biopsies are routinely performed in patients with megaloblastic anemia
to look for any evidence of malabsorption since tropical sprue (TS) is reported to
be more prevalent in India. These biopsies are performed irrespective of the macroscopic
endoscopic findings. The present study was undertaken to study the value of duodenal
biopsy in patients of megaloblastic anemia and correlate the biopsy findings with
endoscopy. As a secondary aim, the study also analyzed the hematological profile of
patients with megaloblastic anemia.
Materials and Methods
Approval was obtained from the institutional human ethics committee before the onset
of the study. All the cases of megaloblastic anemia with bone marrow studies diagnosed
at PSG Institute of Medical Sciences and Research during the period of 2 years from
January 2016 to December 2017 were retrieved. Cases with nonmegaloblastic macrocytic
anemia, those with other associated neoplastic conditions like myelodysplastic syndrome,
and leukemia were excluded from the study. Hematology assays were performed on a LH
780 hematology analyzer (Beckman Coulter, Inc., United States) using volume conductivity
and scatter technology. Clinical and laboratory findings (serum vitamin B12 and folate levels) were retrieved from hospital records of the patients. Duodenal
biopsies of these patients reported in the histopathology department were retrieved.
Endoscopic findings of these cases were retrieved from hospital information system
and tabulated. Histopathological examination was performed after standard processing
and hematoxylin and eosin staining procedures. All the biopsy slides were reviewed
and only those that had well-oriented villi were included. Histomorphological features
like villous and crypt architecture, intraepithelial lymphocyte (IEL) count per hundred
enterocytes, and 20 villous tip enterocytes in 5 villi were studied. Up to 20 IELs
per 100 enterocytes was considered normal; 21 to 30 IELs per 100 enterocytes and more
than 30 per 100 enterocytes were graded as borderline and severe increase, respectively.
Inflammatory cell component in lamina propria—lymphocytes, plasma cells, neutrophils,
eosinophils, and epithelioid histiocytes were also analyzed and the severity was graded
as mild, moderate, and severe.
Only those with unequivocal partial or complete villous atrophy, intraepithelial lymphocytosis,
and increase in inflammatory cell content were diagnosed as TS. Patients with increase
in IELs and no abnormality of villous architecture (Marsh I, lesion) were categorized
as intraepithelial lymphocytosis. Data were analyzed using SPSS software for windows,
version 20.0, IBM Corporation, NY, United States.
Results
There were 93 cases of megaloblastic anemia diagnosed on bone marrow biopsies during
the study period. There were 68 (73.2%) males and 25 (26.8%) females. Age ranged from
16 to 85 years, the median age being 42 years. Out of 93 cases, vitamin B12 assay was done in all whereas folate levels were available in 73.
According to the World Health Organization (WHO), vitamin B12 status in adults is defined by the serum levels of the micronutrient with the following
cutoffs and definitions: greater than 221 pmol/L (> 163 pg/mL) is vitamin “B12 adequacy”; between 148 and 221 pmol/L (109–163 pg/mL) is “low B12”; and lower than 148 pmol/L (< 109 pg/mL) is “B12 deficiency.”[5]
In our series we found 59 (63.4%) out of 93 patients to be vitamin B12 deficient. 18 (19.3%) had low vitamin B12 levels and 16 (17.2%) were categorized as to have adequate B12. Out of the 16 patients with adequate B12, 4 had folate deficiency. Five cases had vitamin B12 levels more than 2,000 pg/mL.
Isolated vitamin B12 deficiency including low levels was present in 45 (48.38%) patients and folate deficiency
(4 ng/mL, reference range being 4.6–34.8 ng/mL) was seen in 4 (4.3%) patients; 32
(34.48%) patients had both vitamin B12 and folate deficiency. Twelve (12.9%) patients had normal serum vitamin B12 and folate levels.
Considering the hematological parameters, mean hemoglobin and mean corpuscular volume
(MCV) of the patients were 6.68 g/dL and 103.9 fL, respectively. MCV was less than
90 fL in five patients (5%). These five patients had coexisting iron deficiency anemia
([Table 1]).
Table 1
Hematological profile of patients with megaloblastic anemia
|
Hematological parameters
|
Mean
|
Range
|
|
Hemoglobin (g/dL)
|
6.2 + 2.37
|
3.0–11.9
|
|
Mean corpuscular volume (fL)
|
103.9 + 15.49
|
51.8–144.4
|
|
Red cell distribution width (%)
|
19.2
|
16–28
|
|
Platelets (×109/L)
|
83.8
|
9–315
|
|
Total leucocyte count (×109/L)
|
3.1
|
1–18.1
|
Fifty-one patients (54.8%) had pancytopenia at presentation (defined by hemoglobin
less than 11 g/dL, total WBC count less than 4 × 109/L and platelet count less than 150 × 109/L), 48 of whom had vitamin B12 levels < 30 pg/mL. Forty patients had bicytopenia and two had anemia. Peripheral
smear and bone marrow morphology are highlighted in [Figs. 1] and [2].
Fig. 1 Peripheral smear with hypersegmented neutrophil and macro-ovalocyte (100×).
Fig. 2 Bone marrow aspirate showing megaloblastic erythroid hyperplasia (100×).
Duodenal biopsies were available for all 93 patients. Out of these, 46 (49.46%) patients
who had villous blunting of any degree (mild, moderate, or severe) and intraepithelial
lymphocytosis in a decrescendo pattern (more IELs in the crypts) and nucleomegaly
were diagnosed as TS ([Figs. 3] and [4]). Five other cases of TS diagnosed previously did not show unequivocal villous blunting
and hence were categorized as intraepithelial lymphocytosis. Sixteen (17.2%) patients
had only intraepithelial lymphocytosis, 16 (17.2%) had peptic duodenitis, and 15 (16.12%)
had no significant pathology ([Fig. 5]).
Fig. 3 Duodenal biopsy with villous blunting (4×, Hematoxylin and Eosin).
Fig. 4 Duodenal biopsy with variable villous blunting (Hematoxylin and Eosin, 4×).
Fig. 5 Duodenal biopsy with marked intraepithelial lymphocytosis (Hematoxylin and Eosin,
40×).
TS was found to be more frequent in the three groups (isolated vitamin B12 deficiency, isolated folate deficiency, and combined B12 and folate deficiency). TS was less frequent in the patients who had normal vitamin
B12 and folate levels ([Table 2]).
Table 2
Duodenal biopsy findings in patients with vitamin B12 and folate deficiency
|
Number of cases (N = 93)
|
Tropical sprue
|
Intraepithelial lymphocytosis
|
Peptic duodenitis
|
No significant pathology
|
|
Vitamin B12 deficiency
n = 45
|
24 (53.3%)
|
8 (17.7%)
|
5 (11.1%)
|
8 (17.7%)
|
|
Folate deficiency
n = 4
|
3 (75%)
|
–
|
1 (25%)
|
–
|
|
Both
n = 32
|
17 (53.1%)
|
6 (18.75%)
|
6 (18.75%)
|
4 (12.5%)
|
|
Normal
n = 12
|
2 (16.6%)
|
2 (16.6%)
|
4 (33.36%)
|
3 (25%)
|
Endoscopic macroscopic changes pertaining to malabsorption like scalloped duodenal
and attenuated duodenal folds were identified in 26.8% (n = 25) of patients. Of these, the biopsy diagnosis of TS was made in 15 patients (60%).
The remaining cases of TS diagnosed on biopsy showed either normal endoscopic appearance
or other features like nodularity ([Table 3]).
Table 3
Endoscopic findings with histological diagnosis
|
N = 93
|
Tropical sprue
(n = 46)
|
Intraepithelial lymphocytosis (n = 16)
|
Peptic duodenitis
(n = 16)
|
No significant pathology
(n = 15)
|
|
Scalloped duodenal folds
n = 25
|
15 (60%)
|
2 (8%)
|
3 (12%)
|
5 (20%)
|
|
Nodularity
n = 25
|
10 (40%)
|
6 (24%)
|
3 (12%)
|
6 (24%)
|
|
Gastroduodenitis
n = 12
|
6 (50%)
|
2 (16.6%)
|
1 (8.33%)
|
3 (25%)
|
|
No macroscopic abnormality
n = 31
|
15 (48.5%)
|
6 (19.3%)
|
8 (25.8%)
|
2 (6.45%)
|
Discussion
Megaloblastic anemia is one of the commonest causes for pancytopenia in India. It
has a characteristic peripheral smear picture (macrocytosis, ovalocytes, and hypersegmented
neutrophils) and low reticulocyte count.[2] Bone marrow examination is done to rule out other neoplastic disorders which can
have megaloblastosis, like myelodysplastic syndrome and leukemia. Serum vitamin B12 and folate levels will point toward the cause for megaloblastic anemia.[6]
[7] Duodenal biopsies are performed to identify if there is any evidence of malabsorption.
The study reports a male predominance with a ratio of 2.7:1 which is considerably
higher when compared with a vast majority of other studies.[8]
[9] The possible explanation could be the higher population of elderly men in the series
in whom vitamin B12 deficiency due to intestinal malabsorption are reported to be common.[10]
Cobalamin and folate deficiency are predominantly due to decreased dietary intake
or malabsorption in Indian population while pernicious anemia has been reported to
be the common etiology in Western population.[11]
[12] Vitamin B12 deficiency has been reported to be the common cause for megaloblastic anemia in our
population constituting to 63%, similar to that of Khandhuri et al (68%)[8] and Sarode et al (76%).[9] Combined vitamin B12 and folate deficiency was seen in 34% of cases which is slightly higher when compared
with 8.8% and 12% reported by Sarode et al and Khandhuri et al, respectively. This
is probably because of higher percentage of people with malabsorption in this series
where combined micronutrient deficiencies are common. Folate deficiency was seen in
4.3% of patients which is similar to other studies. The cases with vitamin B12 levels greater than 2,000 pg/mL could represent cases treated inadvertently with
vitamin B12 injections prior to investigation.
Pancytopenia and bicytopenia were observed in 54.8% and 43% of the patients, respectively.
There are wide variations reported for the frequency of pancytopenia ranging from
17.2 to 43.8%.[13]
[14] Another study done in South India has reported pancytopenia in 40% of the patients.[15] The higher incidence of pancytopenia in our population can be attributed to severe
vitamin B12 deficiency in these patients (48 out of 51 had vitamin B12 levels < 30 pg/mL). Mean MCV observed in the current study was 103.9 + 15.49 fL,
which is lower when compared with that reported by Chidambaram et al (110 fL).[15] MCV was found to be normal in 5.3% of the patients which is lower than another study.[14]
TS was the commonest biopsy diagnosis in the study accounting for 49.5% of patients
diagnosed as having megaloblastic anemia, which is similar to most other studies.[16]
[17] Endoscopic finding of scalloped duodenal folds was identified in 32.6% of TS. In
the remaining cases, the diagnosis was possible only with the help of biopsy.
The diagnostic yield of gastrointestinal endoscopy is low in our study. This could
be because subtle endoscopic changes were not documented since duodenal biopsies were
routinely done in all the patients with megaloblastic anemia. However, Sharma et al[18] in their systematic review had found normal duodenal folds to be more common in
TS.
Intraepithelial lymphocytosis without villous abnormality was the second commonest
biopsy finding identified in 34.7% of patients. IEL without villous abnormality had
been reported in various conditions such as gluten-sensitive enteropathy, infections,
autoimmune conditions, drugs, and idiopathic etiology.[19] It has also been reported to be an early morphological feature of sprue.[19]
[20]
TS and IEL without villous abnormality together constituted 66.6% of cases, indicating
malabsorption as the etiology for vitamin B12 and folate deficiency in these cases. Hence, biopsy contributes in identifying the
etiology in 66.6% of cases.
In the remaining cases, the etiology of megaloblastic anemia could have been dietary
deficiency or intrinsic factor deficiency.
Although the guidelines for investigation of megaloblastic anemia recommends serological
assays of anti-intrinsic factor antibodies (anti-IFAB)[5] the incidence of pernicious anemia is very low in India. Duodenal biopsies have
become a routine practice in the workup more based on the evidence of high number
of cases of TS being reported. Anti-IFAB levels were not done in the patients in the
study.
It was found in the present study that routine duodenal biopsy in cases of megaloblastic
anemia yields etiologic information in majority of the cases, irrespective of endoscopy.
Hence it is recommended even in the absence of macroscopic endoscopic changes.
Current spectrum of malabsorption in India has found celiac sprue to be increasing
in incidence.[19] Since the clinical, endoscopic, and histologic difference between celiac and tropical
sprue are not well defined, serum antibodies against tissue transglutaminase and endomysial
antibodies are needed in cases where there is no response to therapy for TS.
Limitations
Since this is a retrospective study, follow-up of patients to look for response to
therapy for TS is not available. Since the study was conducted in a teaching hospital,
endoscopy being done by different endoscopists would have influenced the results.
Serological tests for celiac sprue and anti-IFAB antibodies were not done in the study.
Conclusion
Pancytopenia was the commonest presentation of megaloblastic anemia; vitamin B12 deficiency and folate deficiency constituted 82% and 4.3%, respectively. The incidence
of TS in megaloblastic anemia is as high as 49.5% in the study population. Duodenal
biopsy is valuable in the workup of megaloblastic anemia, irrespective of the endoscopic
changes in identifying the etiology.
