Analysis of IBA1 Low/Negative Microglia in Liver Disease Patients Protocol
Phase 1: Patient Recruitment and Liver Biopsy Collection (Days 1-30)
Patient Cohort: Recruit adult patients (n=80) undergoing percutaneous liver biopsy for clinical indication at tertiary hepatology center. Include disease groups: (a) non-alcoholic fatty liver disease (NAFLD, n=25), (b) alcoholic liver disease (ALD, n=20), (c) chronic hepatitis B/C (n=20), (d) normal liver histology (controls, n=15). Obtain written informed consent.
Biopsy Processing: Collect research cores (16G needle, ≥1.5 cm length) alongside clinical samples. Immediately snap-freeze in liquid nitrogen for molecular studies and place in RNAlater for RNA preservation. Process remaining tissue in OCT for immunohistochemistry.
...Analysis of IBA1 Low/Negative Microglia in Liver Disease Patients Protocol
Phase 1: Patient Recruitment and Liver Biopsy Collection (Days 1-30)
Patient Cohort: Recruit adult patients (n=80) undergoing percutaneous liver biopsy for clinical indication at tertiary hepatology center. Include disease groups: (a) non-alcoholic fatty liver disease (NAFLD, n=25), (b) alcoholic liver disease (ALD, n=20), (c) chronic hepatitis B/C (n=20), (d) normal liver histology (controls, n=15). Obtain written informed consent.
Biopsy Processing: Collect research cores (16G needle, ≥1.5 cm length) alongside clinical samples. Immediately snap-freeze in liquid nitrogen for molecular studies and place in RNAlater for RNA preservation. Process remaining tissue in OCT for immunohistochemistry.
Clinical Data Collection: Record demographics, BMI, metabolic syndrome criteria, MELD score, Child-Pugh class, liver enzymes (ALT, AST, ALP, GGT), and fibrosis stage (METAVIR, Brunt, or NASH-CRN scoring).
Phase 2: Immunohistochemical Profiling of Macrophage Populations (Days 31-49)
Multiplex Immunofluorescence: Perform 7-color Opal multiplex (PerkinElmer) on frozen liver sections (5 μm). Stain sequence: IBA1 (1:200, Wako #019-19741), CD68 (1:100, Abcam #ab783), CD163 (1:100, Abcam #ab189981, M2 macrophage marker), CD86 (1:50, BD Biosciences #555664, M1 marker), DAPI, and one experimental marker per run. Image on Vectra 3.0.
IBA1 Subset Analysis: Using inForm software, segment liver macrophages as CD68+ cells. Subclassify as IBA1 high (>75th percentile), IBA1 intermediate, or IBA1 low/negative (<25th percentile). Quantify proportion of each subset within total CD68+ macrophages per patient.
Spatial Analysis: Map IBA1 subset distribution relative to: (a) fibrotic septa (collagen I staining), (b) portal tracts, (c) steatotic areas. Calculate proximity (distance to nearest fibrotic strand) for each subset.
Phase 3: Transcriptomic and Functional Characterization (Days 50-70)
qRT-PCR Array: Sort IBA1 high vs. IBA1 low/negative macrophages from fresh liver tissue via magnetic bead separation (CD11b+ enrichment, then FACS based on IBA1 intensity). Extract RNA, run RT² qPCR Array Human Macrophage markers (Qiagen #PAHS-064ZA) covering 84 genes.
Pathway Analysis: Compare gene expression between IBA1 high and IBA1 low subsets. Focus on: (a) M1/M2 polarization markers, (b) fibrogenic genes (COL1A1, ACTA2, TGFB1), (c) inflammatory cytokines (IL1B, IL10, TNF). Calculate fold change and pathway enrichment.
Correlation with Clinical Severity: Correlate IBA1 subset proportions with fibrosis stage (METAVIR F0-F4), NAS score (NAFLD activity score), and clinical liver function tests. Use ordinal logistic regression for fibrosis stage as outcome.