PARR

Echevarne Veterinary offers the PARR technique to improve cancer diagnosis in canine and feline species

What is it?

PARR ("PCR Antigen Receptor Rearrengement") is a technique for the detection of clonality and is useful for distinguishing between lymphoms and inflammatory processes. Lymphomas are clonal expansions derived from a single cell line, while the inflammatory processes are polyclonal.

When is it indicated?

Mostly in cases where the morphology and immunophenotyping are not conclusive. Some situations when it may be indicated:

  • Some forms of low-grade lymphoma, called indolent lymphomas (cases of follicular lymphoma, mantle cell lymphoma).
  • Identification of intestinal lymphomas, cancers that often mimic chronic inflammatory processes. Also useful in distinguishing between thymic lymphoma and thymoma.
  • Identification of Lymphocytosis skin, probably a form of indolent T-cell lymphoma histologically benign.
  • Distinction between leukemia and leukemoid reactions.

What samples must be sent?

The technique allows the use of fresh tissues, tissues fixed in formalin, paraffin samples (biopsies), blood, bone marrow and cytological material.

What sensitivity and specificity it has?

CaninaThe sensitivity of the technique in canine samples is 90% and specificity is 92%*.

FelinaThe sensitivity of the technique in feline samples is 89% and specificity is 96%*

* Both factors depend largely on the type of sample used (fixed or paraffin embedded tissue, fresh tissue) of the primers used for PCR protocol and resolution of the technique of separation of PCR product.

What is it based on?

Lymphomas are haematological tumors caused by clonal expansion of lymphocytes. Clonality detection is based on the fact that lymphocytes contain DNA regions that are unique in length and sequence.

These unique regions are mostly located in genes encoding the CDR3 region (Complementary Determining Region 3) both immunoglobulin and T cell receptor CDR3 is the region that accommodates binding to antigen.

In B cells, CDR3 is produced by the recombination of V genes, D and J, whereas in T cells, recombination occurs between the V and J genes during the recombination process, are added 5 to 20 random nucleotide resulting in the length and sequence diversity. After obtaining genetic material of the sample proceed to PCR amplification using primers specific for the VDJ region (for immunoglobulins) and VJ (for TCR).

Detection of the amplified products is performed by fragmented analysis using an automated sequencer. Polyclonality if a heterogeneous mixture of different chain length is obtained while monoclonality if one peak is obtained corresponding to a single type of channel.


The configuration of genetic loci for immunoglobulin contains 80 V genes, 6 D genes D and 3 to 5 J genes.

During the development of T and B cells, by cleaving mechanisms, and a D and V region are added. In this process there may be added or deleted nucleotides in a  number ranging from one cell to another, which makes each cell different from the others.

The same process is repeated with D and J genes. As a result, we have contiguity between V, D and J genes and a different string length for each cell due to variability in the addition or deletion of nucleotides. When the cell is divided, its offspring inherits the same VDJ configuration. To perform the technique, the genetic material of the sample is isolated. By using primers the VDJ region is amplified:

  • If clonality twin chains are always obtained:


  • If there is polyclonality we obtain an heterogeneous mixture of chains of different lengths:


The results of the different lengths in a graph look like:

REFERENCES

  1. Bao Y, Guo Y, Xiao S, et al. Molecular characterization of the VH repertoire in Canis familiaris. Vet Immunol Immunopathol 2010;137:64–75.
  2. Burnett RC, Vernau W, Modiano JF, et al. Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes. Vet Pathol 2003;40:32–41.
  3. Burnett, R.C., W. Vernau, J.F. Modiano, C.S. Olver, P.F. Moore and A.C. Avery, Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes. Veterinary Pathology, 2003. 40: p. 32-41.
  4. Chaubert P, Baur Chaubert AS, Sattler U, et al. Improved polymerase chain reactionbased method to detect early-stage epitheliotropic T-cell lymphoma (mycosis fungoides) in formalin-fixed, paraffin-embedded skin biopsy specimens of the dog. J Vet Diagn Invest 2010;22:20–9.
  5. Keller RL, Avery AC, Burnett RC, et al. Detection of neoplastic lymphocytes in peripheral blood of dogs with lymphoma by polymerase chain reaction for antigen receptor gene rearrangement. Vet Clin Pathol 2004;33:145–9.
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  7. Moore PF, Woo JC, Vernau W, et al. Characterization of feline T cell receptor gamma (TCRG) variable region genes for the molecular diagnosis of feline intestinal T cell lymphoma. Vet Immunol Immunopathol 2005;106:167–78.
  8. Moore, P.F., J.C. Woo, W. Vernau, S. Kosten and P.S. Graham, Characterization of feline T cell receptor gamma (TCRG) variable region genes for the molecular diagnosis of feline intestinal T cell lymphoma. Veterinary Immunology & Immunopathology, 2005. 106(3-4): p. 167-178.
  9. Tamura K, Yagihara H, Isotani M, et al. Development of the polymerase chain reaction assay based on the canine genome database for detection of monoclonality in B cell lymphoma. Vet Immunol Immunopath 2006;115:163–7.
  10. Vernau W, Moore PF. An immunophenotypic study of canine leukemias and preliminary assessment of clonality by polymerase chain reaction. Vet Immunol Immunopathol 1999;69:145–64.
  11. Weiss AT, Klopfleisch R, Gruber AD. T-cell receptor gamma chain variable and joining region genes of subgroup 1 are clonally rearranged in feline B- and T-cell lymphoma. J Comp Pathol 2011;144:123–34.
  12. Werner, J.A., J.C. Woo, W. Vernau, P.S. Graham, R.A. Grahn, L.A. Lyons and P.F. Moore, Characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of B-cell neoplasia. Veterinary Pathology, 2005. 42(5): p. 596-607.
  13. Yagihara H, Tamura K, Isotania M, et al. Genomic organization of the T-cell receptor Ɣ gene and PCR detection of its clonal rearrangement in canine T-cell lymphoma/leukemia. Vet Immunol Immunopathol 2007;115:375–82.
  14. Yamazaki J, Baba K, Goto-Koshino Y, et al. Quantitative assessment of minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction. Vet Immunol Immunopathol  2008;126:321–31.