Introduction to the Blood Beryllium Lymphocyte Proliferation Test

Chronic beryllium disease (CBD), a disorder mainly affecting the lung, occurs in a small percentage of persons exposed to beryllium dusts. Most investigators require evidence of beryllium hypersensitivity as one of several criteria for diagnosis of the disease . In vitro proliferation of bronchoalveolar lavage cells to beryllium is extremely sensitive to and specific for the diagnosis of CBD but is not suitable for screening since it is an invasive procedure . A noninvasive procedure based on the proliferative response of blood cells to beryllium has been developed and is referred to as the beryllium specific lymphocyte proliferation test (BeLPT). This modification of the standard lymphocyte proliferation test is used to identify relatively rare individuals among worker cohorts who display delayed hypersensitivity reactions when exposed to beryllium metal. The BeLPT involves in vitro challenge of peripheral blood lymphocytes with salts of beryllium combined with assays for clonal proliferation of sensitized subsets of CD4 lymphocytes using tritiated thymidine uptake as a quantitative measure of blastogenesis. The test is conducted using 96-well microtiter plates and the amounts of tritiated thymidine incorporated by replicate wells containing lymphocytes challenged with beryllium is compared with uptake of radioactivity by replicate wells of non-challenged lymphocytes to establish stimulation indices'' (SIs) as a measure of in vitro sensitivity to beryllium. A major problem in the interpretation of BeLPT test results is outlying data values ( about 7%) among the replicate well counts .

The increasing use of beryllium in several new economic sectors emphasizes the need for medical surveillance in the workplace for CBD. In particular, beryllium has been used in the nuclear industry for a number of years. Kreiss et al (1993) have examined the epidemiology of CBD in a stratified sample of workers at a nuclear weapons plant, and discuss the role of the BeLPT in beryllium disease surveillance in the nuclear industry. The U.S. Department of Energy (DOE) is operating a screening program for CBD that will eventually include approximately 15,000 current and former beryllium exposed workers at 20 DOE sites. Each participating beryllium worker will have a BeLPT at an approved laboratory using a standard protocol developed by the Committee to Accredit Beryllium Sensitization Testing (CABST). The results of each assay will then be evaluated and classified as normal, abnormal, or unsatisfactory.

A major concern that was not completely resolved by the CABST was how to deal with outliers'' that occur in the BeLPT data. The main purpose of this report is to propose a new statistical approach that can be used for analysis of a BeLPT assay that may contain multiple outlying well counts. Given their undue influence on the estimates of the SIs, a method for handling outliers is needed. The current approach'' (as described in the July 1993 version of the CABST protocol) is based on an ad hoc outlier rejection method. {\bf As an alternative we propose using resistant estimation methods that are not sensitive to outliers.} The BeLPT assay is described with a regression model that relates the expected well counts at each of the three beryllium concentrations to the control well counts for cells that are harvested after five and seven days. Resistant fitting methods are used to estimate the SI for each of the six beryllium concentrations. The main advantage of this approach is that estimates of the SIs are calculated without explicitly identifying and deleting the outlying well counts.

A second question considered is the identification of beryllium exposed workers who exhibit beryllium hypersensitivity. Most (over 90\%) of the beryllium workers will have SIs similar to those of a control group with no known exposure to beryllium. However, even after the use of resistant estimation methods to minimize the effect of outlying well counts, the BeLPT for some beryllium workers will yield large SIs. In this case we want to identify the outliers'' (i.e. individuals with large SIs), since they represent beryllium workers who exhibit beryllium hypersensitivity.

The Blood Beryllium Lymphocyte Proliferation Test

A detailed description of lymphocyte culture methods, quality control measures, and examples of plate maps and print-outs of raw data are included in the Appendix. Following is a brief description (see Figure) of the protocol for the BeLPT culture assay as established by CABST and implemented by the BeLPT laboratory at Oak Ridge Institute for Science and Education (ORISE) as of July 1993. The details of this procedure and the equipment used vary at different laboratories that are performing the BeLPT.

• A 15 ml blood sample is obtained from each patient and mononuclear cells are separated using density gradient centrifugation.

• Lymphocytes are cultured using standard methods at a final concentration of 2.5 x 10^5 cells per well in 96 well flat bottom microtiter plates. For each BeLPT assay 12 replicate control wells, and four replicates for each experimental condition (i.e., 1, 10, and 100 microM of BeSO4, and mitogen stimulated positive controls) are set up.

• Cells are incubated at 37 degrees C for five and seven days and a pulse of tritiated thymidine is delivered prior to harvest. Cells are harvested on filter paper and counts are measured in a Packard Matrix 96 gas ionization counter. Each filter is counted for thirty minutes and the results organized as shown in Table 1 for statistical analysis.