关键词: 分枝杆菌,结核, 诊断, 病理学, 质量控制, 评价研究

Abstract:

Objective: To develop a standardized method for preparing quality control (QC) paraffin blocks for pathological diagnosis of tuberculosis, and to evaluate their performance with respect to cellular morphology, spatial distribution of Mycobacterium tuberculosis (MTB), and MTB DNA. Methods: QC paraffin blocks containing different concentrations of MTB were prepared by combining inactivated MTB H37Rv suspension with sedimented cells from malignant pleural effusion, using 2% agar as the embedding matrix. Negative control blocks were generated by replacing the MTB suspension with phosphate-buffered saline (PBS). Each cylindrical agar block was sectioned longitudinally into four equal layers (approximately 3 mm thick), yielding stratified sample groups. Hematoxylin and eosin (HE) staining was used to assess the integrity of cellular morphology and the uniformity of spatial distribution. Acid-fast staining (AFB) and quantitative real-time PCR (qPCR) were employed to evaluate MTB load, IS6110 copy number, and the homogeneity of MTB distribution along the longitudinal axis of the blocks. Results: Standardized QC paraffin blocks compatible with both AFB staining and MTB DNA detection were successfully developed. HE staining confirmed the preservation of cellular morphology and a uniform spatial distribution of cells within the blocks. In AFB staining, stratified sample groups containing theoretical MTB loads of 33.6 and 336 bacilli per section consistently exhibited detectable bacilli across all slices, qualifying them as reliable positive QC materials for staining. The corresponding average MTB densities were (21.50±9.26) and (169.25±40.25) bacilli per 300 fields, respectively. Quantitative PCR analysis demonstrated consistent positivity in all blocks with theoretical MTB concentrations of 0.336, 3.36, 33.6, and 336 bacilli per section. The IS6110 gene copy numbers in these groups were (6.48±3.54), (26.53±6.65), (283.93±98.51), and (4446.75±833.84) copies per five sections, respectively. The coefficient of variation for Ct values remained below 5% in all groups, and the longitudinal distribution of MTB DNA within the blocks was uniform, supporting their qualification as standardized molecular QC materials. Negative controls were consistently negative in both AFB staining and qPCR, with no IS6110 amplification observed. Conclusion: We developed a standardized protocol for the preparation of QC materials tailored to the pathological diagnosis of tuberculosis. Based on assay sensitivity and clinical application scenarios, we recommend a minimum MTB load of 33.6 bacilli per section for AFB staining QC and 0.336 bacilli per section for molecular diagnostic QC. The selection of initial MTB concentration should be guided by the detection limits of the assay and the intended diagnostic context.

Key words: Mycobacterium tuberculosis, Diagnosis, Pathology, Quality control, Evaluation studies