Participants
We prospectively enrolled patients aged 1–18 years who underwent HSCT from May 2017 to August 2018 at the Bone Marrow Transplantation Unit, Ramathibodi Hospital, Thailand. Patients were excluded if they had received vitamin A supplementation in the previous 3 months before enrollment or had renal insufficiency (GFR < 50 mL/min), which causes false elevation of citrulline levels [10].
HSCT was performed on the basis of underlying diseases according to our institutional protocols. All participants received conditioning chemotherapy, supportive care and antimicrobial prophylaxis after receiving stem cells.
This trial was approved by the Institutional Ethics Committee, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (ID 03-60-18). All patients and/or their parents provided written informed consent. The study was registered at the Thai Clinical Trials Registry (TCTR20191207002) and partly adhered to CONSORT guidelines.
Study design
This study was a preliminary, single center, prospective, quasi-randomized, clinical trial. Eligible patients were assigned to one of two groups by 1:1 alternation: 15 patients received a single oral dose of 200,000 IU of vitamin A before the conditioning chemotherpay; the other 15 patients did not receive vitamin A supplementation (Supplementary Fig. 1). Baseline characteristics including age, sex, body weight, height, diagnosis, donor type and conditioning chemotherapy regimens were collected.
Blood samples were collected before administration of vitamin A, on stem cell infusion day (day 0), and 7 days after HSCT. Plasma was separated by centrifugation and stored at −80°C until analysis. Plasma retinol and citrulline levels were analyzed using high-performance liquid chromatography (HPLC) and cation-exchange HPLC techniques, respectively.
All of the patients were followed for a period of 60 days after HSCT. Clinical data including number and consistency of stools, fever and oral mucositis were recorded thrice daily (every 8 hours).
Outcomes
The primary outcome of this study was GI mucosal damage after HSCT indicated by plasma citrulline level. Secondary outcomes, assessed up to 60 days after HSCT, were post-HSCT complications including incidence of febrile neutropenia, oral mucositis, infections, diarrhea, graft-versus-host disease (GVHD), hemorrhagic cystitis, time of neutrophil engraftment, length of hospital stay, and mortality. Incidence of vitamin A toxicity (headache, vomiting, seizure, change in mental status, visual disturbance) was also recorded.
Definitions
Diarrhea was defined as passage of ≥ 3 watery stools in a 24-hour period. Bacterial infection was defined as presence of pathogenic bacteria in cultures of blood, cerebrospinal fluid or > 105 CFU/mL in cultures of urine. Pneumonia was diagnosed by imaging study. Febrile neutropenia was defined as a single oral temperature measurement > 38.3°C, or a temperature > 38°C sustained over a 1-hour period with absolute neutrophil count < 500 cells/µL or expected to decrease to < 500 cells/ µL during the next 48 hours. Neutrophil engraftment was defined as an absolute neutrophil count > 500 cells/ µL for 3 consecutive days after HSCT. Vitamin A deficiency and insufficiency were defined as plasma vitamin A levels < 20 and 30 µg/dL, respectively.
Statistical analysis
Between-group comparisons were analyzed using the Mann Whitney U-test or the Student’s t-test for continuous data, and the χ2 test or Fisher’s exact test for categorical data. Comparisons of plasma vitamin A and citrulline levels between the two groups were performed by mixed linear regression. Statistical significance was defined as a p value < 0.05. Statistical analysis of data was performed using the Stata version 15.1 (StataCorp LLC, College Station, TX, USA).