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

Aires Lobo^*, Narciss Okhravi^*, Peter Adamson, Brian J Clark and Susan Lightman^*

From the Departments of Clinical Ophthalmology, * Cell Biology, and Pathology, Institute of Ophthalmology, Moorfields Eye Hospital, London; and Pharmagene Laboratories Limited, Royston, United Kingdom

To determine the usefulness of polymerase chain reaction (PCR)analyses in the diagnosis of lymphoid infiltrate cells in ocularsamples, PCR was performed using oligonucleotide primers specificfor immunoglobulin heavy chain rearrangement at framework 2,framework 3, and t(14;18) translocation of the bcl-2 gene. Thesewere 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-celllymphoma DNA. PCR against Fr2 and Fr3 IgH rearrangement detected10 fg and 10 pg in the seminested PCR, respectively. Conventionalpathological methods were highly accurate at establishing thecorrect final diagnosis in formalin-fixed, paraffin-embeddedsamples but were much less sensitive and predictive in cytologicalspecimens of intraocular fluid. A combination of the three PCRreactions was an equally successful diagnostic approach on paraffin-embeddedsamples, whereas single PCR reactions did not significantlyimprove diagnosis over histopathological diagnostic techniques.Thus, a combination of PCR reactions is useful in the detectionof B-cell monoclonality, aids the differentiation between lymphomatousand inflammatory infiltrates, and is more powerful as a diagnosticmethod than single PCR or conventional cytopathology for lymphoidinfiltrates in ocular fluid aspirates.