Detection of neoplastic lymphocytes in peripheral blood of dogs with lymphoma by polymerase chain reaction for antigen receptor gene rearrangement

Background: Uniquely rearranged immunoglobulin and T‐cell receptor gene sequences can be amplified and electrophoretically separated by size to detect a clonal population of lymphocytes. Objective: The purpose of this study was to determine whether the polymerase chain reaction (PCR) detects neoplastic (clonal) lymphocytes more frequently than do microscopic methods. Methods: We identified neoplastic lymphocytes in peripheral blood by both routine and standardized microscopic examination of blood smears and by PCR amplification of blood‐derived DNA and compared the 3 methods for frequency of detection of leukemic involvement. For standardized microscopic examination (200 leukocytes counted on Wright‐Giemsa‐stained blood smears), samples were categorized as negative (≤1% prolymphocytes), equivocal (>1% prolymphocytes, no lymphoblasts), or positive (≥1 lymphoblast). A PCR‐amplified sample was positive if 1 or 2 discrete bands were seen on the gel, or negative if no bands, a smear, or a faint ladder was seen. Results: Using PCR, neoplastic lymphocytes were detected in peripheral blood 2.5 times more frequently than with routine or standardized microscopic evaluation. Eighty‐three percent of samples negative by microscopy were positive by PCR. Conclusion: PCR is more sensitive than microscopy for the detection of clonal lymphocytes in peripheral blood. The results of this study also suggest that neoplastic lymphocytes circulate in peripheral blood at a higher frequency than previously reported. PCR may be useful for detecting or phenotyping lymphoma, monitoring response to therapy, identifying recurrence, and screening breeds at risk.