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Abstract
PCR for antigen receptor gene rearrangements (PARR) analysis is being increasingly used to assist diagnosis of canine lymphoma. In this study, PARR was carried out on consecutive samples received as part of routine diagnostic practice from 271 patients: 195 with lymphoid malignancies, 53 with reactive conditions and 23 with other neoplasms. Initially, published primer sets were used but later minor primer modifications were introduced and primers were rationalised to give a PARR panel that provides a good compromise between sensitivity and cost. Results were compared to diagnoses made by histology or cytology, coupled with immunophenotyping by flow cytometry or immunohistochemistry where possible. After exclusion of 11 poor quality samples, 230/260 (88%) gave a clear result with 162/163 (99%) of samples classified as clonal and 56/67 (84%) classified as polyclonal giving results concordant with the cytological/histological diagnosis. Among 30 samples with equivocal results, 21 had clonal peaks in a polyclonal background and nine showed little amplification. These were from patients with a range of neoplastic and non-neoplastic conditions emphasising the need to interpret such results carefully in concert with other diagnostic tests. The combination of primer sets used in this study resulted in a robust, highly specific and sensitive assay for detecting clonality.
Highlights
PCR for antigen receptor gene rearrangements (PARR) is increasingly being used in the diagnostic setting as a means of assessing clonality in samples with a differential diagnosis of canine lymphoma
Using the published primer sets in their original pairings (Tables S1 and S2), the majority of samples diagnosed as B-cell lymphoma had detectable clonal immunoglobulin heavy chain (IgH) rearrangements (73 of 81; 90.1%; see Table 6)
A smaller proportion of the T-cell lymphomas had identifiable clonal TCR␥ rearrangements (29 of 39; 74.4%), and only 72.9% (35 of 48) of lymphoma not otherwise specified’ (NOS) samples had a detectable clone. Since these findings suggested that some rearrangements, of TCR␥, were being missed a systematic review of the primer sets was undertaken to try and improve sensitivity, as described in Materials and Methods
Summary
PCR for antigen receptor gene rearrangements (PARR) is increasingly being used in the diagnostic setting as a means of assessing clonality in samples with a differential diagnosis of canine lymphoma. Following publication of the canine genome (Lindblad-Toh et al, 2005), primers were modified and added to provide better gene coverage and improve sensitivity (Tamura et al, 2006; Yagihara et al, 2007; Chaubert et al, 2010); full annotations of the TCR␥ and IgH loci were not published until 2009 and 2010, respectively (Massari et al, 2009; Bao et al, 2010) Studies prior to this did not consider all described segments, with only a single publication using a comprehensive PCR strategy addressing all known gene segments, and this only for TCR␥ (Keller and Moore, 2012). It is unlikely that currently used primer sets will detect all antigen receptor gene rearrangements, resulting in sub-optimal sensitivity
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