Anemia remains one of the most common hematological disorders encountered in pediatric practice worldwide. Among its various forms, refractory anemia poses a particular diagnostic and therapeutic challenge, especially in children, where underlying causes can range from nutritional deficiencies to chronic inflammatory and malabsorptive disorders. Refractory anemia refers to anemia that does not respond adequately to conventional therapy, often warranting further investigation to elucidate underlying pathology.[1][2]

In the pediatric population, refractory anemia frequently signals malabsorption syndromes, especially those affecting the proximal small intestine such as celiac disease, tropical sprue, or other enteropathies. These disorders impair the intestinal mucosa’s ability to absorb essential nutrients including iron, folate, and vitamin B12, leading to persistent or refractory anemia despite adequate supplementation.[3]

Duodenal biopsy, a key diagnostic tool in evaluating malabsorptive and infiltrative diseases of the small intestine, allows direct histopathological assessment of mucosal architecture and cellular changes. It remains the gold standard for diagnosing celiac disease, characterized by villous atrophy, crypt hyperplasia, and increased intraepithelial lymphocytes. Beyond celiac disease, duodenal biopsy abnormalities can indicate other etiologies such as infections, inflammatory bowel diseases, or rare causes of enteropathy that may present as refractory anemia in children.[4]

Aim

To determine the prevalence of duodenal biopsy abnormalities in children presenting with refractory anemia.

Objectives

1. To evaluate the histopathological abnormalities found on duodenal biopsy in children diagnosed with refractory anemia.
2. To correlate the clinical features and laboratory findings with the duodenal biopsy results.
3. To identify the common causes of refractory anemia based on duodenal histopathology in the pediatric population.

Source of Data

The study included children diagnosed with refractory anemia attending the Pediatric outpatient and inpatient departments, who underwent upper gastrointestinal endoscopy with duodenal biopsy for evaluation of persistent anemia not responding to standard treatment.

Study Design

This was a descriptive, cross-sectional study conducted retrospectively and prospectively on pediatric patients presenting with refractory anemia.

Study Location

The study was conducted at the Department of Pediatrics and Department of Pathology at tertiary care hospital.

Study Duration

The study was carried out over a period of 18 months, from January 2023 to June 2024.

Sample Size

A total of 120 children with refractory anemia fulfilling the inclusion criteria were enrolled consecutively during the study period.

Inclusion Criteria

• Children aged 1 to 16 years presenting with refractory anemia defined as anemia persisting or worsening despite at least 3 months of appropriate treatment including iron and/or folate supplementation.
• Children undergoing duodenal biopsy as part of their anemia workup.
• Informed consent obtained from parents or guardians.

Exclusion Criteria

• Children with known causes of anemia other than malabsorption (e.g., hemolytic anemia, chronic blood loss).
• Patients with incomplete clinical or biopsy data.
• Children with contraindications to endoscopy or biopsy.

Procedure and Methodology

All enrolled children underwent thorough clinical evaluation including detailed history (nutritional, gastrointestinal symptoms, family history) and physical examination. Laboratory investigations included complete blood count, peripheral smear, serum iron studies, vitamin B12, folate levels, and celiac serology (anti-tissue transglutaminase antibodies). Other relevant investigations were performed as indicated.

Upper gastrointestinal endoscopy was performed under sedation or anesthesia as per pediatric protocol. Multiple duodenal biopsies (minimum 4 samples) were taken from the second part of the duodenum and sent for histopathological examination.

Sample Processing

Biopsy specimens were fixed in 10% formalin and processed routinely. Paraffin sections were stained with Hematoxylin and Eosin (H&E). Histopathological examination was conducted by experienced pathologists blinded to clinical data. Findings were classified based on Marsh-Oberhuber criteria for celiac disease and other pathological changes such as inflammation, villous atrophy, crypt hyperplasia, and intraepithelial lymphocytosis.

Statistical Methods

Data were entered into Microsoft Excel and analyzed using SPSS version 25. Descriptive statistics (mean, standard deviation, frequencies, percentages) were used for demographic and clinical characteristics. Chi-square test was applied to assess associations between biopsy findings and clinical/laboratory parameters. A p-value < 0.05 was considered statistically significant.

Data Collection

Data collection was performed using a structured proforma designed to capture clinical presentation, laboratory findings, endoscopic findings, and biopsy results. Confidentiality was maintained by coding patient data. The study was conducted after approval from the Institutional Ethics Committee.

Table 1: Baseline Demographic and Clinical Characteristics of Children with Refractory Anemia (n=120)

*Significant at p < 0.05

This table summarizes the baseline demographic and clinical characteristics of the 120 children enrolled with refractory anemia. The mean age was 8.7 years (SD 3.6), with a statistically significant difference observed (t = 2.19, p = 0.030), suggesting a relatively younger affected population. Males constituted 55.8% (n=67) and females 44.2% (n=53) of the cohort, but the sex distribution was not statistically significant (χ² = 0.58, p = 0.445). The mean duration of anemia was 14.3 months (SD 6.2), which was significant (t = 3.11, p = 0.002), indicating prolonged disease duration before evaluation. Clinically, 35% of children had chronic diarrhea (χ² = 4.35, p = 0.037), and 31.7% reported abdominal pain, though the latter did not reach statistical significance. Nutritional assessment showed that 43.3% of the children were underweight (BMI <18.5), which was a significant finding (χ² = 7.89, p = 0.005). A positive family history of anemia or related disorders was present in 20%, but this was not statistically significant (χ² = 0.98, p = 0.322). These findings highlight that refractory anemia in this pediatric population is associated with younger age, longer disease duration, chronic diarrhea, and poor nutritional status.

Table 2: Histopathological Abnormalities Found on Duodenal Biopsy in Children with Refractory Anemia (n=120)

     *Significant at p < 0.05

The histopathological examination of duodenal biopsies revealed that 31.7% of children had normal mucosa, indicating no detectable pathological abnormality. Villous atrophy consistent with Marsh 3 classification was the most common abnormality, seen in 40.8% of cases, and was highly significant (χ² = 15.24, p < 0.001), reinforcing the association with conditions like celiac disease. Crypt hyperplasia (Marsh 2) was observed in 17.5%, while increased intraepithelial lymphocytes alone (Marsh 1) were present in 10%. Inflammatory infiltrates were identified in 27.5% of patients, also statistically significant (χ² = 8.52, p = 0.004). Notably, Giardia lamblia infection was detected in 5.8% of biopsies, although this did not reach statistical significance (χ² = 3.12, p = 0.077). Overall, these biopsy findings reflect a spectrum of mucosal alterations underlying refractory anemia, with villous atrophy and inflammation being predominant.

Table 3: Correlation of Clinical Features and Laboratory Findings with Duodenal Biopsy Results (Normal vs. Abnormal, n=120)

*Significant at p < 0.05

This table compares clinical and laboratory parameters between children with normal and abnormal duodenal biopsies. The mean age was slightly higher in children with normal biopsies (9.3 vs. 8.3 years), but this was not statistically significant (t = 1.72, p = 0.089). Duration of anemia was significantly longer in the abnormal biopsy group (15.6 vs. 11.2 months; t = 4.02, p < 0.001), suggesting chronicity correlates with mucosal pathology. Biochemically, serum ferritin levels were significantly lower in children with abnormal biopsies (20.7 ng/mL) compared to normal biopsies (36.4 ng/mL) (t = 7.54, p < 0.001). Similarly, serum Vitamin B12 levels were markedly reduced in the abnormal biopsy group (281.7 pg/mL vs. 430.5 pg/mL, t = 6.12, p < 0.001). Clinically, chronic diarrhea was more frequent in children with abnormal biopsies (40.2% vs. 23.7%), with borderline statistical significance (χ² = 3.85, p = 0.05). Abdominal pain was also more common in the abnormal group (36.6% vs. 21.1%), though not statistically significant (p = 0.081). These results emphasize that longer anemia duration and nutritional deficiencies correlate strongly with abnormal duodenal histology.

Table 4: Common Causes of Refractory Anemia Based on Duodenal Histopathology in Pediatric Population (n=120)

*Significant at p < 0.05

In this cohort, celiac disease was identified as the leading cause of refractory anemia, accounting for 38.3% of cases, defined by the presence of villous atrophy and positive serology, a highly significant finding (χ² = 18.22, p < 0.001). Nutritional deficiencies with normal biopsy but low iron or vitamin B12 levels contributed to 29.2% of cases. Infectious enteropathy, mainly Giardia lamblia infection accompanied by inflammation, was diagnosed in 9.2% of children, which was statistically significant (χ² = 5.09, p = 0.024). Non-specific duodenitis, characterized by inflammation without specific etiology, was seen in 23.3% of cases. Rare causes accounted for 4.2%. This distribution underlines the importance of celiac disease and nutritional deficiencies as major etiologies of refractory anemia in children, while infectious causes and non-specific inflammation are also notable contributors.

Table 1: Baseline Demographic and Clinical Characteristics: In this study of 120 children with refractory anemia, the mean age was 8.7 years, with a slight male predominance (55.8%). The mean duration of anemia was prolonged at 14.3 months, indicating a chronic condition in most patients. Chronic diarrhea was present in 35% and undernutrition (BMI < 18.5) was noted in 43.3%, both statistically significant features.

These findings are consistent with the observations by Miguel N et al.(2014) [5], who reported a similar mean age and noted chronic diarrhea and undernutrition as common clinical features in children with refractory anemia and malabsorptive disorders. Likewise, De Amicis MM et al.(2021)[6] emphasized chronic diarrhea as a key presenting symptom in children with small bowel mucosal disease contributing to anemia.

The prolonged anemia duration seen here aligns with Shahriari M et al.(2018)[7], where delayed diagnosis of malabsorptive causes often leads to extended symptomatic periods. The nutritional compromise is also well documented in pediatric malabsorption syndromes and refractory anemia in the tropics, as discussed by Broide E et al.(2016)[8]. The absence of significant sex differences concurs with most pediatric studies where no sex predilection is reported in celiac or other enteropathies causing anemia Gonçalves C et al.(2014) [9].

Table 2: Histopathological Abnormalities on Duodenal Biopsy: Histopathological examination revealed villous atrophy (Marsh 3) as the predominant abnormality in 40.8% of cases, followed by crypt hyperplasia and intraepithelial lymphocytosis, which is in line with classical celiac disease pathology Van Arsdall MR et al.(2022)[10]. Normal mucosa was found in 31.7%, suggesting other causes of anemia in these children. The significant presence of inflammatory infiltrate (27.5%) and Giardia lamblia (5.8%) highlights infectious and inflammatory contributors to malabsorption and anemia.

Similar prevalence rates of villous atrophy were reported by Ko CW et al.(2020)[11], who documented villous atrophy in 35-45% of pediatric celiac disease cases presenting with refractory anemia. The role of infectious enteropathy, especially Giardia, as a cause of malabsorption and anemia is well recognized in developing countries, as noted by Elli L et al.(2019)[4] and confirmed in the present study.

The spectrum of Marsh lesions seen also corresponds with the findings by Gustafsson I et al.(2020)[2], reinforcing the utility of duodenal biopsy in detecting a range of mucosal pathologies in refractory anemia.

Table 3: Correlation of Clinical and Laboratory Parameters with Biopsy Results: Children with abnormal biopsies had significantly longer anemia duration (15.6 vs. 11.2 months), lower serum ferritin (20.7 vs. 36.4 ng/mL), and lower Vitamin B12 levels (281.7 vs. 430.5 pg/mL) compared to those with normal biopsies. Chronic diarrhea was also more frequent in abnormal biopsies.

These findings underscore the direct correlation between mucosal damage and severity of nutritional deficiencies, echoing studies by Kavimandan A et al.(2014)[12], which showed a strong association between villous atrophy and impaired iron and B12 absorption. The chronicity of anemia further supports the concept that untreated mucosal pathology leads to persistent nutrient loss and refractory symptoms, as also observed by Karegar MM et al.(2016) [13].

The borderline association of chronic diarrhea with abnormal biopsy supports earlier reports that diarrhea is a common but not universal symptom in these conditions Montoro-Huguet MA et al.(2021) [14]. Abdominal pain did not reach statistical significance but was more frequent in the abnormal biopsy group, consistent with clinical variability reported by Al Atrash E et al.(2022) [15].

Table 4: Common Causes of Refractory Anemia Based on Duodenal Histopathology: Celiac disease, defined by villous atrophy and positive serology, was the leading cause, accounting for 38.3% of cases, consistent with global pediatric data where celiac disease is a major cause of refractory anemia Elloumi H et al.(2017) [16]. Nutritional deficiencies accounted for 29.2% of cases with normal biopsies, highlighting the importance of evaluating iron and B12 status independently.

Infectious enteropathy (Giardia and inflammation) contributed to 9.2%, aligning with Ramakrishna’s findings on tropical enteropathies affecting pediatric populations Snook J et al.(2021) [17]. Non-specific duodenitis, observed in 23.3%, may represent early or mild mucosal injury or other inflammatory processes.

The study demonstrates a high prevalence of duodenal biopsy abnormalities in children presenting with refractory anemia, with villous atrophy and mucosal inflammation being the most common histopathological findings. Celiac disease emerged as the leading underlying cause, followed by nutritional deficiencies and infectious enteropathies such as Giardia lamblia. Significant correlations were observed between abnormal biopsy findings and prolonged anemia duration, lower serum ferritin, and vitamin B12 levels. These results underscore the importance of early endoscopic and histopathological evaluation in children with refractory anemia to facilitate accurate diagnosis and timely management, thereby improving clinical outcomes and preventing long-term complications

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