DUAL DETERMINATION OF HELPER AND CYTOTOXIC PRECURSOR LYMPHOCYTE FREQUENCY
USING A MINIATURIZED VITAL DYE-RELEASE METHOD.
Nancy Hensel, Vaishali Agarwala, Yin-Zheng Jiang, A. John Barrett.
Bone Marrow Transplant Unit, Hematology Branch, NHLBI, National Institutes
of Health, Bethesda, MD .
The measurement of helper (HTLP) and cytotoxic (CTLP)
lymphocyte precursor frequencies has the potential for compatibility testing
in bone marrow and organ transplants. Classic methods are limited by the
need for large numbers of stimulator and responder cells and the use of
3H and 51Cr. We developed a split HTLP/CTLP assay using Terasaki plates
(40µl/well) to test lymphocytes in a limiting dilution assay(LDA)
from 3x104 to 0.1x104 responders/well with 3x104 stimulators/well in the
unrelated setting. Supernatant for the HTLP is removed on D+3; IL-2 is
added to the CTLP on D+3 and D+7. Cytotoxicity is tested on D+10 by adding
103/well calcein-AM (CAM) dye-labelled targets (PHAB). Lysis is measured
by a decrease in the fluorescent intensity of each well. An IL-2 dependent
cell line (9.12) suspension is added to the HTLP. After incubation, CAM
is added to the trays and taken up by the IL-2 dependent cells. Proliferation
of the cell line is directly related to the amount of IL-2 per well. The
fluorescent intensity is measured using the Lambda Scan(tm) PlusII/Fluoro
Module attached to an inverted fluorescent microscope. Alternatively, the
fluorescence can be manually visualized and graded in a manner similar
to analyzing the standard lymphocyte cytotoxicity. The entire LDA (HTLP+CTLP;
24 replicates/dilution) requires only 6x106 stimulators, 5x106 responders
and 2.4x105 targets. Results are comparable with conventional assays. The
dual CTLP/HTLP assay distinguishes between HLA mismatched and identical
pairs. The ease of testing and the reduced cell requirement make possible
an extension of the the useful application of the assay.