The accurate distinction of reactive and neoplastic lymphoid proliferations can present challenges. Given the different prognoses and treatment strategies, a correct diagnosis is crucial. Molecular clonality assays assess rearranged lymphocyte antigen receptor gene diversity and can help differentiate reactive from neoplastic lymphoid proliferations. Molecular clonality assays are commonly used to assess atypical, mixed, or mature lymphoid proliferations; small tissue fragments that lack architecture; and fluid samples. In addition, clonality testing can be utilized to track neoplastic clones over time or across anatomic sites. Molecular clonality assays are not stand-alone tests but useful adjuncts that follow clinical, morphologic, and immunophenotypic assessment. Even though clonality testing provides valuable information in a variety of situations, the complexities and pitfalls of this method, as well as its dependency on the experience of the interpreter, are often understated. In addition, a lack of standardized terminology, laboratory practices, and interpretational guidelines hinders the reproducibility of clonality testing across laboratories in veterinary medicine. The objectives of this review are twofold. First, the review is intended to familiarize the diagnostic pathologist or interested clinician with the concepts, potential pitfalls, and limitations of clonality testing. Second, the review strives to provide a basis for future harmonization of clonality testing in veterinary medicine by providing diagnostic guidelines.

https://journals.sagepub.com/doi/pdf/10.1177/0300985815626576

Clonality Testing in Veterinary Medicine: A Review With Diagnostic Guidelines

Effectiveness of Capillary Electrophoresis Using Fluorescent-Labeled Primers in Detecting T-Cell Receptor γ Gene Rearrangements

Context Classic Hodgkin lymphoma (cHL) is regarded as a clonal B-cell neoplasm. The BIOMED-2 group recently validated a set of immunoglobulin heavy chain (IGH) multiplex primers with high sensitivity in B-cell non-Hodgkin lymphomas. We postulated that after using these primers, a higher proportion of the cHLs would have detectable rearrangements without microdissection. Design Forty-two patients with cHL were selected. The densities of Reed-Sternberg cells/10 high-power field and CD30+ cells/10 high-power field were classified as low, intermediate, or high. The quantities of background CD20+ B cells were classified as low or high. DNA from formalin-fixed, paraffin-embedded sections was used to perform polymerase chain reactions with the InVivoScribe IGH Gene Clonality Assay for ABI detection. Dominant peaks were considered to be monoclonal if they were >3x the height of the polyclonal background, and borderline monoclonal if between 2 and 3x. Result Overall, 10/42 (24%) of the cHL samples were monoclonal, and 7/42 (17%) were borderline monoclonal. Higher densities of CD30+ cells and lower background B cells were statistically correlated with clonality. Conclusions The BIOMED-2 primers demonstrate IGH gene clonality in 24% to 40% of cHLs without microdissection. In a subset of the cHL, the IGH gene rearrangement analysis might be useful for diagnosis, but can lead to confusion between cHLs and non-Hodgkin lymphomas if used as a discriminative criterion.

Detection of immunoglobulin heavy chain gene rearrangements in classic hodgkin lymphoma using commercially available BIOMED-2 primers.

Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR

A Novel Method for Interpretation of T-Cell Receptor γ Gene Rearrangement Assay by Capillary Gel Electrophoresis Based on Normal Distribution

Polymerase chain reaction (PCR) technique is widely used in the diagnosis of lymphoma, and PCR amplification products are typically detected by polyacrylamide gel electrophoresis (PAGE). However, the identification of small clonal populations, or the distinction of clonal PCR products in a polyclonal milieu remains difficult, requiring technically demanding alterations to gel analysis. This study describes an alternative approach using a capillary electrophoresis (CE) system to produce an accurately sized electropherogram. A variety of patient samples were examined, including solid tissue, peripheral blood, bone marrow aspirates, and paraffin-embedded tissue. A total of 28 samples were evaluated by PCR for B-cell clonality by detection of immunoglobulin heavy chain gene rearrangement and 29 samples for T-cell clonality by detection of T-cell gamma locus gene rearrangement. Standard 10% PAGE analysis of PCR products was compared with CE. There was a 100% concordance in the assessment of both B-cell and T-cell clonality. Dilution studies with the SUP-B15 cell line showed a detection limit of 0.03% for B-cell clonality and 0.05% for T-cell clonality using CE, versus 0.2% to 1%, respectively for PAGE. Automated, fluorescent analysis of PCR products by CE seems to be at least equally as effective as gel-based analysis for the detection of clonal B-cell and T-cell populations. Moreover. CE offers superior resolution and improved sensitivity, thus representing a significant improvement over traditional gel electrophoretic techniques in these regards.

Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction.

Molecular diagnosis in pediatric acute leukemias.

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Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction.

Molecular diagnosis in pediatric acute leukemias.