Method Description

CD3+ T cells are enriched and purified from EDTA whole blood. RNA is obtained from the T cells and converted into complementary DNA (cDNA) to maximize sample stability. The cDNA is amplified using the polymerase chain reaction (PCR), during which the different CDR3 fragment lengths of the T-cell receptor (TCR) V beta families are fluorescently labeled. The pool of varying CDR3 fragment lengths is separated by size on a capillary electrophoresis genetic analyzer. As the fluorescent label of each CDR3 fragment passes through the laser, the size and fluorescent intensity is recorded. The resulting image is a cluster of fluorescent peaks with single-base-pair separation and different fluorescent intensities, approximately corresponding to the number of fragments of that size represented in the patient's original RNA. The peak patterns are reviewed for organization (number of peaks), relative intensity across peaks, and size distribution. The number of individual peaks is compared to a reference range established from over 140 healthy donors equally represented by both sexes and across the pediatric and adult age spectrum. The reporting units are normalized among the patient population by using a diversity ratio for each TCR V beta family. The diversity ratio for each V beta family is determined by the number of peaks in that specific family relative to all peaks within the patient's sample expressed as a percentage. The analytical process in the laboratory utilizes a variety of controls to assess the performance of the assay and reliability of the result provided. The fragment length analysis is performed by the Gene Marker software and the spectratype is assembled for interpretation. An interpretive report will be provided for each patient sample and includes information on the diversity ratio for each family. The spectratype will be made available on request of the physician.(Unpublished Mayo method)