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Detection of beryllium sensitivity using a flow cytometric lymphocyte proliferation test: the Immuno-Be-LPT
G M Farris1, L S Newman, E L Frome
1Cytometry Group, BioScience Division, Los Alamos National Laboratory, NM 87545, USA.
Insights
A new flow cytometry method improves beryllium hypersensitivity testing by identifying specific T-cell subsets. This Immuno-Be-LPT assay offers insights into chronic beryllium disease (CBD) diagnosis and pathogenesis.
Area of Science:
- Immunology
- Cell Biology
- Toxicology
Background:
- Current beryllium lymphocyte proliferation testing (Be-LPT) uses tritiated thymidine incorporation, lacking cell viability and subset information.
- Chronic beryllium disease (CBD) diagnosis relies on accurate assessment of beryllium-specific immune responses.
- Flow cytometry offers potential for more detailed analysis of lymphocyte proliferation.
Purpose of the Study:
- To develop and validate flow cytometric assays (Immuno-Be-LPT) for measuring beryllium-induced lymphocyte proliferation.
- To compare the Immuno-Be-LPT with the traditional Be-LPT.
- To identify specific lymphocyte subsets involved in beryllium hypersensitivity and CBD.
Main Methods:
- Development of flow cytometric assays combining immunophenotyping with bromodeoxyuridine (BrdU) incorporation or DNA content analysis (PI, DAPI).
- Evaluation of beryllium-induced lymphocyte proliferation in blood samples from CBD patients and beryllium workers using both Be-LPT and Immuno-Be-LPT.
- Analysis of responding lymphocyte subsets (CD4+, CD8+) in beryllium-exposed individuals.
Main Results:
- The Immuno-Be-LPT showed agreement with the traditional Be-LPT.
- Immuno-Be-LPT provided detailed information on proliferating lymphocyte subsets.
- CD4+ lymphocytes proliferated in all CBD patients; CD8+ cells proliferated in most CBD patients and some beryllium workers without CBD.
- Individual's own plasma enhanced CD4+ T-cell proliferation compared to commercial serum.
Conclusions:
- The Immuno-Be-LPT is a reliable method for assessing beryllium-induced lymphocyte proliferation.
- CD4+ lymphocyte response in Immuno-Be-LPT may serve as a novel diagnostic marker for CBD.
- This assay enhances understanding of immune responses in beryllium exposure and CBD pathogenesis.
Abstract:
Measurement of lymphocyte proliferation to detect hypersensitivity to beryllium (Be-LPT) in vitro is done presently using a method based on tritiated thymidine incorporation. Although this method is sensitive it gives no information on cell viability or responding lymphocyte subsets. We have developed reliable and simple flow cytometric assays for lymphocyte proliferation testing (Immuno-Be-LPT) by combining immunophenotyping with bromodeoxyuridine (BrdU) incorporation or DNA content using propidium iodide (PI) or 4'6'-diimidazolin-2-phenylindole (DAPI). Evaluation of beryllium-induced lymphocyte proliferation in blood cells from seven patients with chronic beryllium disease (CBD) and 120 beryllium workers by both the Bc-LPT and the Immuno-Be-LPT showed agreement between the tests. The Immuno-Bc-LPT provided additional information about the specific type of lymphocytes responding. CD4+ lymphocytes proliferated in response to beryllium in blood samples from all seven CBD individuals and CD8+ lymphocytes proliferated in six of the seven. Four beryllium workers without CBD had positive responses to beryllium primarily in the CD8+ cells. The use of the individual's own plasma supported a greater beryllium or tetanus-induced proliferation of CD4+ lymphocytes when compared to commercial human serum. The response of CD4+ lymphocytes measured in the Immuno-Be-LPT may provide a new marker for the diagnosis of CBD.
