Review for 10.1186/s12876-025-03995-9 (2025)

Title: CD45+ erythroid progenitor cells as potential biomarkers for disease progression in
hepatitis B virus-related acute-on-chronic liver failure
Version: 0 Date: 05 Apr 2025
Reviewer's report:
Major Concerns
1. Sample Size and Overfitting in Multivariate Analysis
- The study includes 65 HBV-ACLF patients, split into regression (n=32) and deterioration
(n=33) groups. While the total sample size is modest, the multivariate logistic regression
model initially incorporated 13 variables (Table 4), raising concerns about overfitting. Although
stepwise regression reduced the final model to 2 predictors (INR and NLR), the wide confidence
interval for INR (OR: 17.47, 95% CI: 2.50–121.92) indicates imprecision and instability in effect
estimates. A validation cohort is absent, further limiting reliability.
2. Lack of Adjustment for Confounders
- Age significantly differed between CHB and ACLF groups (39.67 vs. 47.05 years, *p* <
0.001), but it was not included in the regression model. Age-related immune or metabolic
changes could confound associations between CD45# EPCs and outcomes. Other potential
confounders (e.g., treatment regimens, comorbidities) were not addressed.
3. Retrospective Design and Selection Bias
- The retrospective nature introduces risks of unmeasured confounding. No flow diagram or
detailed exclusion criteria (e.g., number of excluded patients) are provided in the main text,
obscuring selection bias. Prospective validation is needed to confirm findings.
4. Small Sample for Immunofluorescence
- Liver tissue analysis included only 3 CHB and 3 ACLF patients, with qualitative
immunofluorescence data. This small sample lacks statistical power and quantitative validation
(e.g., cell counts), weakening conclusions about hepatic CD45# EPC accumulation.
5. Mechanistic Speculation Without Experimental Validation
- The discussion proposes mechanisms (e.g., arginase-2, PD-L1) based on prior literature but
does not experimentally validate these pathways in the study cohort. This limits the paper’s
contribution to mechanistic understanding.
Minor Concerns
1. Inconsistent Reporting
- Discrepancies in decimal places for *p*-values (e.g., Table 1: *p* = 0.031 vs. Table 2: *p* =
0.026) and cutoff values (1.016% in Table 3 vs. 1.02% in the text) need standardization.
2. Weak Clinical Correlations
- Spearman correlations between CD45# EPCs and complications (e.g., HE: *r* = 0.247, *p*
= 0.047; disease progression: *r* = 0.348, *p* = 0.005) are statistically significant but weak,
raising questions about clinical relevance.
3. Figure and Table Clarity
- Figure 1a lacks representative flow cytometry plots, hindering reproducibility assessment.
Table 4 omits confidence intervals for NLR (reported as 1.05–2.89 but not tabulated).
Abbreviations (e.g., MDSCs) are defined late in the text.
4. Reference Errors
- Reference 8 (Engelmann et al.) is cited as 2025, likely a typo. Other citations (e.g., Ref. 36:
Elahi et al. 2013 vs. Ref. 38: Sarin et al. 2014) should be cross-checked for accuracy.
Limitations Not Adequately Addressed
1. Overfitting Risk in Regression
- The authors acknowledge the small sample size but do not explicitly discuss overfitting risks
from stepwise regression or the instability of ORs (e.g., INR’s wide CI).
2. Qualitative Immunofluorescence Data
- The limitations section mentions the lack of single-cell RNA sequencing but does not address
the qualitative nature and minimal sample size of liver tissue analyses.
3. Short Follow-Up and Long-Term Outcomes
- While the 28-day follow-up is noted as a limitation, the clinical significance of CD45# EPCs
for long-term outcomes (e.g., 90-day mortality, HCC risk) remains unexplored.
4. Treatment Heterogeneity
- All patients received nucleoside analogs, but variability in treatment duration or adjunct
therapies (e.g., immunosuppressants) is not discussed, potentially influencing outcomes.