Evaluating Overweight and Obese Children for Suspected NAFLD
Screening for NAFLD and referral to paediatric gastroenterology were performed clinically in primary care prior to participation in the study. Eligibility for this study was designed to mirror the paediatric screening guidelines for NAFLD. Therefore, children had to be at least 10 years old and either overweight or obese. In the clinical notes from the primary care office, there had to be documentation of screening for NAFLD with ALT and referral to paediatric gastroenterology for evaluation of 'elevated ALT' or 'suspected NAFLD' or 'NAFLD'. There was no study inclusion threshold set for ALT; rather, the determination that the screening ALT was abnormal was made by the primary care provider. The parent(s) of all subjects provided written informed consent. Written assent was obtained for all children. The protocol was approved by the institutional review boards of the University of California, San Diego and Rady Children's Hospital San Diego.
The clinical evaluation of the children was at the discretion of the attending paediatric gastroenterologist and not dictated by research protocol. Information generated in the clinical evaluation of suspected NAFLD was captured prospectively. To provide clinical context for the study, details of this diagnostic process are provided.
Paediatric Gastroenterology Clinic. All children underwent a comprehensive history and physical as part of the clinical consultation for suspected NAFLD. The history included investigation of potential hepatotoxic medication intake as well as age-appropriate interviewing for relevant lifestyle factors including unprotected sexual activity, alcohol, tobacco and recreational drug use. Height and weight were measured. Physical examination also included assessment for signs of chronic liver disease. Initial laboratory studies performed in all children at least 1 month after screening labs included hepatic panel (ALT, AST, alkaline phosphatase, total protein, albumin, total bilirubin, direct bilirubin) and gamma glutamyl transferase (GGT) to assess the chronicity and nature of aminotransferase elevation; complete blood count to assess for anaemia and evidence of splenic sequestration related to portal hypertension; and coagulation studies to assess hepatic synthetic function. If this confirmatory testing showed continued evidence for liver disease, additional laboratory studies were performed as ordered by the paediatric gastroenterologist based on the clinical context. These labs included evaluation of both hepatic aetiologies [hepatitis A IgM, hepatitis B surface antigen, hepatitis B surface antibody, hepatitis C antibody, HIV ELISA, alpha-1 anti-trypsin phenotype, anti-nuclear antibody (ANA), anti-smooth muscle antibody (ASMA), anti-liver kidney microsomal antibody, quantitative IgG, ceruloplasmin, 24-h urinary copper measurement] and extra-hepatic aetiologies (tissue transglutaminase IgA, quantitative IgA, serum amino acids, urine organic acids, serum acylcarnitine, profile, creatine kinase, erythrocyte sedimentation rate, C-reactive protein, thyroid stimulating hormone and free thyroxine). In children who had no symptoms or signs of liver disease by history or physical examination, and all laboratory results were normal on confirmatory testing, further evaluation was not pursued, given low likelihood of liver disease. When there was evidence for chronic liver disease based on history, physical and/or laboratory testing, a percutaneous liver biopsy was offered for diagnosis.
Liver Biopsy. Children who underwent clinical liver biopsy did so according to the standard clinical protocol in use at our institution. Anaesthesia was provided by an attending paediatric anaesthesiologist. Children underwent a mask induction using a combination of gas ventilation with oxygen, nitrous oxide and sevoflurane. An intravenous line was placed and supplemental propofol and fentanyl was given according to the anaesthesiologist's discretion. Hemodynamic monitoring was placed. Most patients were mask ventilated or had laryngeal mask airways placed. Ventilation was spontaneous. A time out was performed to confirm the correct patient and procedure. Children were positioned supine, with the right hand raised. Limited ultrasonography of the liver was performed to identify the ideal biopsy path. Liver biopsy was performed by an experienced paediatric gastroenterologist using a 15-gauge Jamshidi needle. A portion of tissue was placed in saline and brought afresh to pathology and a portion was placed in formalin for standard processing. After the procedure was completed, patients were taken to the post-anaesthesia care unit (PACU), monitored and observed for 4 h prior to discharge.
Pathology. Pathology procedures were performed according to standard clinical protocol within our institution. Formalin-fixed paraffin-embedded liver sections were stained with haematoxylin and eosin, periodic acid Schiff with and without diastase, Masson trichrome, reticulin and iron histochemical stains. Fresh frozen sections were stained with oil-red-O. Slides were evaluated systematically (adequacy, overall architecture, portal tracts, and parenchyma) by an experienced, board-certified, paediatric pathologist. Additional stains were performed and reviewed as needed based on the clinical context. Fibrosis was staged using standard methods relevant to the specific pathologic findings (e.g. Kleiner for NAFLD, METAVIR for viral hepatitis, etc.). The pathological diagnosis was recorded.
Diagnosis. The final diagnosis was made by the paediatric gastroenterologist incorporating all available information from clinical interview, medical record, physical examination, laboratory testing and review of histopathology. A diagnosis of NAFLD was based on exclusion of other causes of steatosis by clinical history, laboratory studies and histology in addition to histologic demonstration of ≥5% of hepatocytes containing macrovesicular fat. Following the prevailing standard, for those biopsies indicative of NAFLD, the diagnosis of steatohepatitis was based on the pathologists' interpretation of the global histological features including steatosis, lobular and portal inflammation and ballooning degeneration of hepatocytes. The diagnoses of other liver diseases were made based on relevant society guidelines and standard gastroenterology, hepatology and pathology reference textbooks. For example, the diagnosis of autoimmune hepatitis was made following the recommendations of the AASLD Practice Guideline on the Diagnosis and Management of Autoimmune Hepatitis.
Information generated in the clinical evaluation of suspected NAFLD was captured prospectively. Age and sex were recorded. Because race and ethnicity influence the risk for NAFLD, each child's race and ethnicity were self-identified by the parent(s). Height and weight were recorded. From the primary care provider's office records, the screening ALT and AST values were recorded. In addition, from the paediatric gastroenterology clinical record, we prospectively recorded the values for laboratory assays performed for the evaluation of liver disease. Adverse events were recorded as problems in the operating room, patient complaints in the PACU, return to the hospital, calls to gastroenterology and by report at out-patient follow-up visit.
Calculated Variables. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. BMI percentiles were determined from the Centers for Disease Control and Prevention 2000 growth curves. BMI Z scores (s.d.s from the national reference mean for a given age and gender of children's BMI values) also were determined.
Definitions. Subjects were classified as overweight (BMI 85–94th percentiles) or obese (BMI ≥95th percentile). The most common value used at children's hospitals in the United States for the ULN for ALT is 40 U/L. Therefore, two times the ULN was defined as 80 U/L. This value was used to assess the diagnostic performance of the paediatric guideline recommendation to use two times the ULN for ALT as the threshold for further evaluation. As a comparison, we also evaluated recently proposed biology-based thresholds for the ULN in children derived from the SAFETY study. These are gender-specific with ULN of 25 U/L in boys and 22 U/L in girls. Therefore, two times the biology-based ULN was defined as 50 U/L for boys and 44 U/L for girls. Advanced fibrosis was defined as bridging fibrosis or cirrhosis.
Statistics. Data were expressed as mean ± standard deviation (if not normally distributed, then geometric means were reported) or frequency and percentage. Continuous variables were analysed with Student's t-test; the Mann–Whitney U test was used for nonparametric measures. The Pearson χ test was used to test for differences in proportions. All hypothesis tests were two-tailed. Significance was defined a priori at α value of 0.05. Analyses were performed with Statistica 10 (StatSoft, Inc., Tulsa, OK, USA).
We performed a post-hoc analysis of published indices for NAFLD and advanced fibrosis using readily available clinical data points. For the detection of NAFLD, we tested the Fatty Liver Index (FLI). The FLI was calculated as (e*loge (triglycerides) + 0.139*BMI + 0.718*loge (GGT) + 0.053*waist circumference–15.745)/(1 + e*loge (triglycerides) + 0.139*BMI + 0.718*loge (GGT) + 0.053*waist circumference–15.745) × 100. The index produces a score from 1 to 100. Scores of <30 are considered negative for NAFLD, scores between 31 and 59 are considered indeterminate and scores >60 are considered positive for NAFLD. For the detection of advanced fibrosis, we tested the FIB-4 index. This was calculated as (Age × AST)/(Platelets × (sqr (ALT)). Values <1.3 are considered negative, values between 1.3 and 2.67 are considered indeterminate and values ≥2.67 are considered positive for advanced fibrosis. Finally, we tested the Paediatric NAFLD Fibrosis Index (PNFI). A linear predictor for PNFI was calculated as linear predictor (lp) = –6.539 × loge [age (years)] + 0.207 × waist (cm) + 1.957 × loge [triglycerides (mg/dl)] – 10.074. This linear predictor is transformed into a PNFI score: (1/1 + e) × 10. PNFI scores ≥9 are considered positive for fibrosis. We tested for differences in the PNFI score between those children with and without advanced fibrosis.
Methods
Study Population
Screening for NAFLD and referral to paediatric gastroenterology were performed clinically in primary care prior to participation in the study. Eligibility for this study was designed to mirror the paediatric screening guidelines for NAFLD. Therefore, children had to be at least 10 years old and either overweight or obese. In the clinical notes from the primary care office, there had to be documentation of screening for NAFLD with ALT and referral to paediatric gastroenterology for evaluation of 'elevated ALT' or 'suspected NAFLD' or 'NAFLD'. There was no study inclusion threshold set for ALT; rather, the determination that the screening ALT was abnormal was made by the primary care provider. The parent(s) of all subjects provided written informed consent. Written assent was obtained for all children. The protocol was approved by the institutional review boards of the University of California, San Diego and Rady Children's Hospital San Diego.
Standard of Care Clinical Evaluation of Suspected NAFLD
The clinical evaluation of the children was at the discretion of the attending paediatric gastroenterologist and not dictated by research protocol. Information generated in the clinical evaluation of suspected NAFLD was captured prospectively. To provide clinical context for the study, details of this diagnostic process are provided.
Paediatric Gastroenterology Clinic. All children underwent a comprehensive history and physical as part of the clinical consultation for suspected NAFLD. The history included investigation of potential hepatotoxic medication intake as well as age-appropriate interviewing for relevant lifestyle factors including unprotected sexual activity, alcohol, tobacco and recreational drug use. Height and weight were measured. Physical examination also included assessment for signs of chronic liver disease. Initial laboratory studies performed in all children at least 1 month after screening labs included hepatic panel (ALT, AST, alkaline phosphatase, total protein, albumin, total bilirubin, direct bilirubin) and gamma glutamyl transferase (GGT) to assess the chronicity and nature of aminotransferase elevation; complete blood count to assess for anaemia and evidence of splenic sequestration related to portal hypertension; and coagulation studies to assess hepatic synthetic function. If this confirmatory testing showed continued evidence for liver disease, additional laboratory studies were performed as ordered by the paediatric gastroenterologist based on the clinical context. These labs included evaluation of both hepatic aetiologies [hepatitis A IgM, hepatitis B surface antigen, hepatitis B surface antibody, hepatitis C antibody, HIV ELISA, alpha-1 anti-trypsin phenotype, anti-nuclear antibody (ANA), anti-smooth muscle antibody (ASMA), anti-liver kidney microsomal antibody, quantitative IgG, ceruloplasmin, 24-h urinary copper measurement] and extra-hepatic aetiologies (tissue transglutaminase IgA, quantitative IgA, serum amino acids, urine organic acids, serum acylcarnitine, profile, creatine kinase, erythrocyte sedimentation rate, C-reactive protein, thyroid stimulating hormone and free thyroxine). In children who had no symptoms or signs of liver disease by history or physical examination, and all laboratory results were normal on confirmatory testing, further evaluation was not pursued, given low likelihood of liver disease. When there was evidence for chronic liver disease based on history, physical and/or laboratory testing, a percutaneous liver biopsy was offered for diagnosis.
Liver Biopsy. Children who underwent clinical liver biopsy did so according to the standard clinical protocol in use at our institution. Anaesthesia was provided by an attending paediatric anaesthesiologist. Children underwent a mask induction using a combination of gas ventilation with oxygen, nitrous oxide and sevoflurane. An intravenous line was placed and supplemental propofol and fentanyl was given according to the anaesthesiologist's discretion. Hemodynamic monitoring was placed. Most patients were mask ventilated or had laryngeal mask airways placed. Ventilation was spontaneous. A time out was performed to confirm the correct patient and procedure. Children were positioned supine, with the right hand raised. Limited ultrasonography of the liver was performed to identify the ideal biopsy path. Liver biopsy was performed by an experienced paediatric gastroenterologist using a 15-gauge Jamshidi needle. A portion of tissue was placed in saline and brought afresh to pathology and a portion was placed in formalin for standard processing. After the procedure was completed, patients were taken to the post-anaesthesia care unit (PACU), monitored and observed for 4 h prior to discharge.
Pathology. Pathology procedures were performed according to standard clinical protocol within our institution. Formalin-fixed paraffin-embedded liver sections were stained with haematoxylin and eosin, periodic acid Schiff with and without diastase, Masson trichrome, reticulin and iron histochemical stains. Fresh frozen sections were stained with oil-red-O. Slides were evaluated systematically (adequacy, overall architecture, portal tracts, and parenchyma) by an experienced, board-certified, paediatric pathologist. Additional stains were performed and reviewed as needed based on the clinical context. Fibrosis was staged using standard methods relevant to the specific pathologic findings (e.g. Kleiner for NAFLD, METAVIR for viral hepatitis, etc.). The pathological diagnosis was recorded.
Diagnosis. The final diagnosis was made by the paediatric gastroenterologist incorporating all available information from clinical interview, medical record, physical examination, laboratory testing and review of histopathology. A diagnosis of NAFLD was based on exclusion of other causes of steatosis by clinical history, laboratory studies and histology in addition to histologic demonstration of ≥5% of hepatocytes containing macrovesicular fat. Following the prevailing standard, for those biopsies indicative of NAFLD, the diagnosis of steatohepatitis was based on the pathologists' interpretation of the global histological features including steatosis, lobular and portal inflammation and ballooning degeneration of hepatocytes. The diagnoses of other liver diseases were made based on relevant society guidelines and standard gastroenterology, hepatology and pathology reference textbooks. For example, the diagnosis of autoimmune hepatitis was made following the recommendations of the AASLD Practice Guideline on the Diagnosis and Management of Autoimmune Hepatitis.
Data Collection
Information generated in the clinical evaluation of suspected NAFLD was captured prospectively. Age and sex were recorded. Because race and ethnicity influence the risk for NAFLD, each child's race and ethnicity were self-identified by the parent(s). Height and weight were recorded. From the primary care provider's office records, the screening ALT and AST values were recorded. In addition, from the paediatric gastroenterology clinical record, we prospectively recorded the values for laboratory assays performed for the evaluation of liver disease. Adverse events were recorded as problems in the operating room, patient complaints in the PACU, return to the hospital, calls to gastroenterology and by report at out-patient follow-up visit.
Data Analysis
Calculated Variables. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. BMI percentiles were determined from the Centers for Disease Control and Prevention 2000 growth curves. BMI Z scores (s.d.s from the national reference mean for a given age and gender of children's BMI values) also were determined.
Definitions. Subjects were classified as overweight (BMI 85–94th percentiles) or obese (BMI ≥95th percentile). The most common value used at children's hospitals in the United States for the ULN for ALT is 40 U/L. Therefore, two times the ULN was defined as 80 U/L. This value was used to assess the diagnostic performance of the paediatric guideline recommendation to use two times the ULN for ALT as the threshold for further evaluation. As a comparison, we also evaluated recently proposed biology-based thresholds for the ULN in children derived from the SAFETY study. These are gender-specific with ULN of 25 U/L in boys and 22 U/L in girls. Therefore, two times the biology-based ULN was defined as 50 U/L for boys and 44 U/L for girls. Advanced fibrosis was defined as bridging fibrosis or cirrhosis.
Statistics. Data were expressed as mean ± standard deviation (if not normally distributed, then geometric means were reported) or frequency and percentage. Continuous variables were analysed with Student's t-test; the Mann–Whitney U test was used for nonparametric measures. The Pearson χ test was used to test for differences in proportions. All hypothesis tests were two-tailed. Significance was defined a priori at α value of 0.05. Analyses were performed with Statistica 10 (StatSoft, Inc., Tulsa, OK, USA).
We performed a post-hoc analysis of published indices for NAFLD and advanced fibrosis using readily available clinical data points. For the detection of NAFLD, we tested the Fatty Liver Index (FLI). The FLI was calculated as (e*loge (triglycerides) + 0.139*BMI + 0.718*loge (GGT) + 0.053*waist circumference–15.745)/(1 + e*loge (triglycerides) + 0.139*BMI + 0.718*loge (GGT) + 0.053*waist circumference–15.745) × 100. The index produces a score from 1 to 100. Scores of <30 are considered negative for NAFLD, scores between 31 and 59 are considered indeterminate and scores >60 are considered positive for NAFLD. For the detection of advanced fibrosis, we tested the FIB-4 index. This was calculated as (Age × AST)/(Platelets × (sqr (ALT)). Values <1.3 are considered negative, values between 1.3 and 2.67 are considered indeterminate and values ≥2.67 are considered positive for advanced fibrosis. Finally, we tested the Paediatric NAFLD Fibrosis Index (PNFI). A linear predictor for PNFI was calculated as linear predictor (lp) = –6.539 × loge [age (years)] + 0.207 × waist (cm) + 1.957 × loge [triglycerides (mg/dl)] – 10.074. This linear predictor is transformed into a PNFI score: (1/1 + e) × 10. PNFI scores ≥9 are considered positive for fibrosis. We tested for differences in the PNFI score between those children with and without advanced fibrosis.
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