Protocol for the Use of Polymerase Chain Reaction in the Detection of Intraocular Large B-Cell Lymphoma in Ocular Samples

Abstract

To determine the usefulness of polymerase chain reaction (PCR) analyses in the diagnosis of lymphoid infiltrate cells in ocular samples, PCR was performed using oligonucleotide primers specific for immunoglobulin heavy chain rearrangement at framework 2, framework 3, and t(14;18) translocation of the bcl-2 gene. These were used to successfully generate amplicons of 220 to 230 bp, 110 to 120 bp, and 175 to 200 bp, respectively. After PCR amplification, primers directed against the t(14;18) detected 10 pg of B-cell lymphoma DNA. PCR against Fr2 and Fr3 IgH rearrangement detected 10 fg and 10 pg in the seminested PCR, respectively. Conventional pathological methods were highly accurate at establishing the correct final diagnosis in formalin-fixed, paraffin-embedded samples but were much less sensitive and predictive in cytological specimens of intraocular fluid. A combination of the three PCR reactions was an equally successful diagnostic approach on paraffin-embedded samples, whereas single PCR reactions did not significantly improve diagnosis over histopathological diagnostic techniques. Thus, a combination of PCR reactions is useful in the detection of B-cell monoclonality, aids the differentiation between lymphomatous and inflammatory infiltrates, and is more powerful as a diagnostic method than single PCR or conventional cytopathology for lymphoid infiltrates in ocular fluid aspirates.

The pathologist's perspective on vitreous opacities

“…Should sufficient material be available for examination, investigation of the vitreous specimens for rearrangements of the immunoglobulin heavy chain gene using PCR provides further evidence of the neoplastic nature of the lymphocytic infiltrate in retinal lymphoma. 17,18,20,22,25,35,49,51 Biochemical analysis of the vitreous specimen for interleukin ratios (IL10 : IL6) may also support the diagnosis of retinal lymphoma. [52][53][54] If possible, retinal lymphoma should be distinguished from other types of intraocular lymphoma, namely primary uveal lymphoma and secondary (metastatic) intraocular lymphoma.…”

Emerging diagnostic tests for vitreoretinal lymphoma: a review

“…In 2007, Lobo et al . from Moorfields Eye Hospital performed two PCR analyses with different primer sets on 28 paraffin‐embedded material or fresh vitreous from cases with a differential diagnosis that included vitreoretinal lymphoma, plus normal vitreous taken at macular hole surgery: a clonal rearrangement was detected in 16 lymphomas with early histopathological confirmation, and importantly, in four additional lymphomas that were initially undiagnosed and recognized only in follow‐up. With both primer sets, sensitivity was 0.95 – higher than cytopathology – and specificity was 1.00.…”

“…However, there are important considerations for the design of tests and interpretation of results, including reduced sensitivity associated with a polyclonal background, and reduced specificity when small numbers of cells in a sample result in a pseudoclonality pattern; thus analyses should be duplicated and multiple targets should be included. 11 The first application of the technology to vitreoretinal lymphoma was by Katai et 20 performed two PCR analyses with different primer sets on 28 paraffin-embedded material or fresh vitreous from cases with a differential diagnosis that included vitreoretinal lymphoma, plus normal vitreous taken at macular hole surgery: a clonal rearrangement was detected in 16 lymphomas with early histopathological confirmation, and importantly, in four additional lymphomas that were initially undiagnosed and recognized only in followup. With both primer sets, sensitivity was 0.95higher than cytopathologyand specificity was 1.00.…”

“…In a second larger study from the NIH group, 25 t(14:18) was identified in 41 of 72 (57%) cytologically confirmed vitreoretinal lymphoma cases, using vitreous material that was microdissected from Giemsa-stained slides. Lobo et al 20 studied their 28 cases with differential diagnosis of vitreoretinal lymphoma for t (14:18), alongside the IGH gene rearrangement, and identified the translocation in 4 of 20 tumour cases. Notably, their comparison of the sensitivity and specificity of different combinations of PCR-based diagnostics indicated that PCR for t(14:18) had little impact on accuracy.…”

Primary Intraocular Lymphoma

“…150 Mmltiplex primers and semi-nested PCR increase the chances of primer annealing, thus enhancing the detection of monoclonal population. 79,140,198 PCR based studies have suggested that the B-cells of PIOL are mature B-cells that have undergone the germinal center reaction and the identical clone can be identified in ocular and cerebral tissues in cases of CNS involvement. 55,57,141 IgH gene rearrangements are a marker of clonality, rather than malignancy, and have been found in samples with nonneoplastic uveitis and may be absent in samples diagnosed as PIOL by cytology and flow cytometry.…”