Graft-versus-host disease represents a frequent complication following allogeneic hematopoietic stem cell transplantation, characterized by a rapid and potentially life-threatening course. Current diagnostic approaches for graft-versus-host disease rely on the recognition of nonspecific clinical signs and the use of invasive procedures (biopsy). The field is actively investigating alternative approaches, including the use of soluble biomarkers. Within a multicenter study framework, two promising biomarkers were identified: regenerating islet-derived 3-alpha and soluble suppressor of tumorigenicity 2. The combination of regenerating islet-derived 3-alpha and soluble suppressor of tumorigenicity 2 demonstrates high predictive power for graft-versus-host disease and enables early diagnosis and treatment monitoring when used in combination. This study presents the validation process for methods used to determine serum concentrations of regenerating islet-derived 3-alpha and soluble suppressor of tumorigenicity 2 using solid-phase enzyme-linked immunosorbent assay. Validation followed recommendations from the Slovenian Association for Clinical Chemistry and Laboratory Medicine for analytical procedure and instrument verification in clinical laboratories. To determine the serum concentration of regenerating islet-derived 3-alpha, the Ab-Match Assembly Human PAP1 (REG3α) kit, combined with the Ab-Match Universal kit (MBL International Corporation) was used. For quantification of soluble ST2, the DuoSet ELISA Development System Human ST2/IL-33R (DY523B-05) and DuoSet Ancillary Reagent Kit 2 (Bio-Techne R&D Systems) were used. Based on measured absorbance, we assessed linearity, intra-assay and inter-assay precision, and accuracy. We evaluated the work according to predetermined criteria. We also evaluated possible interferences (hemolysis, lipemia, autoimmune hemolytic anemia, immune thrombocytopenia). Both methods demonstrated acceptable linearity, precision (intra- and inter-assay), and accuracy. The aforementioned interferences proved to be clinically insignificant. This thesis confirms the clinical utility of serum quantification of regenerating islet-derived 3-alpha and soluble suppressor of tumorigenicity 2 within a predictive model for acute graft-versus-host disease probability assessment. Such biomarker determination represents a noninvasive alternative to current diagnostic approaches. The thesis presents temporal biomarker profiles in patients with and without graft-versus-host disease, along with their interpretation relative to the clinical disease course and treatment response. Statistical analysis of 31 patient samples revealed statistically significant differences in regenerating islet-derived 3-alpha and soluble suppressor of tumorigenicity 2 concentrations between graft-versus-host disease and non-graft-versus-host disease patients (p < 0.05). Future studies should determine regenerating islet-derived 3-alpha and soluble suppressor of tumorigenicity 2 serum concentrations in patients with conditions (inflammatory diseases, gastrointestinal disorders, autoimmune diseases) that may cause nonspecific biomarker elevation, which would contribute to a more accurate interpretation of results. Given the statistical analysis of only 31 patient samples, study expansion is recommended.
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