T Cell Receptor (TCR) V Repertoire Clonality Screening in Lung Transplant Bronchoalveolar Lavage (BAL) Specimens Using a Simple One Step PCR Method
RW Allan, SM Shunkwiler, ZC Kou, CS Dadisman, IM Bovio, MA Baz. Univ. Florida, Gainesville, FL; BioMed Immunotech, Tampa, FL
Background: Clinical management of lung transplant patients requires accurate, timely diagnosis and treatment of rejection. Transbronchial biopsies are performed for the monitoring of rejection though the method is invasive and is associated with complications. BAL is routinely performed at bronchoscopy on transplant patients. BAL fluid contains abundant graft infiltrating lymphocytes; therefore this may be monitored for the development of allograft reactive T cells. We sought to determine if a simple, clinically applicable assay, SuperTCRExpresstm TCR clonality detection [S-TCREx] (BioMed Immunotech, FL), could be used to diagnose rejection by identifying restricted TCR V usage in BAL lymphocytes. S-TCREx uses a one step-PCR and a simple agarose gel based assay to determine clonality of the 22 individual Vb families.
Design: Five lung transplant patients were selected at three time points with differing degrees of rejection at transbronchial biopsy. A total of 15 BAL samples were retrieved from a frozen bank (5 patients x 3 time points) and analyzed using the S-TCREx method and visualized on 4% high resolution agarose gels. For each sample the individual V family usage was scored as: no amplified signal, clonal (1 discreet band), oligoclonal (>1 discreet band), or polyclonal (ill defined band).
Results: S-TCREx yielded amplified bands from frozen banked BAL samples in all samples. The total number of amplified V families ranged from 3 to 20, in 3/5 patients cellular rejection was associated with a restricted V repertoire (<10 amplified families). In all samples there was a range of clonal (5%-67%), oligoclonal (17-75%) and polyclonal (0-70%) V families between patients (Fig 1).
Conclusions: Restricted V repertoire and clonal/oligoclonal T cell expansion within V families are common in transplant BAL samples. At at cutoff of 50% of the amplified V families showing clonal bands, there was an association with clinically significant acute cellular rejection (A2) (p=0.01, Fisher exact test, Sensitivity 75%, Specificity 100%) using ROC analysis.
Monday, March 9, 2009 1:00 PM
Poster Session II # 237, Monday Afternoon