Project Identifier: LLNL-90-103
Project Title:
Somatic Cell Mutations Detected in Human Reticulocytes
Principle Investigator:
Dr. Richard G. Langlois
Project started in: 1990
Project Funding Information:
Project received funding in Fiscal Year 1995.
Project used human subjects in Fiscal Year 1995.
Funding Sources:
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: January 18, 1995
IRB Approval Number: 90-103
Number of Human Subjects in the Last Reporting Period for this Project: 25
(Reporting periods vary.)
Type of Human Subjects Involvement:
A. Objectives
Blood samples will be used for the development and application of a new assay for somatic cell mutations at the glycophorin A (GPA) locus detected in human reticulocytes. A reticulocyte-based mutagenesis assay has a number of potential advantages over our current erythrocyte-based GPA assay. These include a rapid response and increased sensitivity to acute exposures to genotoxic agents, and the potential for molecular characterization of the mutations responsible for the variant phenotype reticulocytes detected by the assay. Thus, the reticulocyte project should provide important new capabilities for studying the human risk from exposure to genotoxic agents and for studying the molecular nature of fetal mutations.
B. Methodology
Blood samples (5-15 ml) from volunteers at LLNL will be obtained by standard venipuncture done by members of the LLNL Medical Department. Similar samples of cord blood collected by outside collaborators will be refrigerated and sent to LLNL within one week of collection. All samples will first be M,N typed to identify which samples are of blood type MN, as the assay can only be performed on samples of this blood type. Centrifugation or immunomagnetic selection procedures will then be used to obtain samples enriched in reticulocytes. The enriched samples will be fixed, labeled with monoclonal antibodies and propidium iodide, and analyzed by flow cytometry. The flow cytometer data will be used to calculate the frequency of variant phenotype reticulocytes in each sample. For some samples, flow sorting will be used to purify variant reticulocytes for molecular analysis of messenger RNA in these cells.
C. Ionizing Radiation, Radioactive Substances, or Chemical Substances
None
D. Involvement of Human Subjects
Blood samples will be used for the development and application of a new assay for somatic cell mutations at the glycophorin A (GPA) locus detected in human reticulocytes. Blood samples from LLNL volunteers and cord blood samples will be used for the development of this new assay system. After the assay is developed, it will be applied to samples from unexposed individuals and from individuals that have been exposed to ionizing radiation or mutagenic chemicals. These samples will be provided by outside collaborators that have access to populations of exposed individuals.
Samples will be obtained from two sources. Samples from unexposed volunteers at LLNL are currently being collected for the erythrocyte-based GPA assay (IRB# 91-102, R. Langlois, P.I.). Samples of cord blood will be obtained from The Children's Hospital, Denver, CO.
Samples from outside collaborations will be encoded by the collaborator, so the names of the participants will not be known by anyone at LLNL. Samples from LLNL volunteers will be encoded by the Principal Investigator, Dr. Richard Langlois, and all data and records will use code numbers only.
Blood samples are obtained by standard venipuncture procedures, and it is expected that the subject will be able to function normally immediately. Possible risks and discomforts that may result from the procedure are considered unlikely but include:
a. Temporary pain
b. Bruising and/or soreness of the affected tissue or surrounding tissue
c. Formation of scar tissue
d. Infection
e. Fainting