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.
