Characterization of bone marrow CD4 to CD8 ratios and lymphocyte composition in adults by image analysis

Bone marrow (BM) lymphocyte subsets are evaluated by flow cytometry or immunohistochemistry for diagnostic purposes; however, CD4:CD8 T lymphocyte ratios are often erroneously interpreted using peripheral blood ranges. There are few and no recent studies describing the composition of lymphocytes within the marrow space, or normal reference ranges. Lymphocyte subsets in cytopenic patients and hospital autopsy BM specimens were evaluated to better characterize CD4:CD8 ratios. Ten patients with a history of cytopenia were identified from 2017 to 2021. Clinical history, cytogenetic testing, and results of a next generation sequencing panel were reviewed to rule out hematolymphoid disease. Thirty-five decedents who underwent a hospital autopsy from 2018 to 2019 were identified. History of hematolymphoid disease was ruled out by chart review. Immunohistochemical staining for CD3, CD20, CD4, and CD8 was evaluated with digital image analysis. Findings were compared to peripheral blood flow cytometry in a group of 20 living patients. BM CD4:CD8 ratios by image analysis were significantly lower than peripheral blood, mean in cytopenic patients 0.37:1 and mean in decedents 0.51:1 versus 2.6:1 (p = <.001 in both groups). BM CD4:CD8 ratios were significantly lower (p = 0.04) than ratios found using flow cytometry on the same specimen, suggesting hemodilution. There was no significant difference in CD4:CD8 ratios when comparing living patients and decedents’ marrows (p = > 0.99). Lymphoid aggregates were encountered with increasing frequency in older individuals. These findings aid in the evaluation of BM lymphocyte subsets and distribution both in living patients and autopsy evaluation. We also present a practical approach to image analysis.