Molecular pathology of primary intraocular lymphoma

Biopsy techniques and yields in diagnosing primary intraocular lymphoma

“…Indeed, at the NEI we are familiar with cytologic evaluation being noncontributory in making a diagnosis of PIOL. While we frequently employ molecular pathologic analysis (microdissection and PCR) to determine monoclonal rearrangement of the IgH gene [24,25,29] this test is still considered to be adjunctive to cytology. Appropriate time points to perform PCR are shown in Fig.…”

“…At the NEI we use primers including those for the second framework region (FR2A 5′-TGGRTCCGMCAGSCVYCNGG-3′), the third frame work region (FR3A 5 ′-A C A -CGGCYSTGTATTACTGT-3′), and CDR3 (5′-CCGGRAARRGTCTGGAGTGG-3′) to detect monoclonality within the variable region of the third complementary determining region (CDR3) in the IgH gene (Fig. 4B) [24,25,29]. Very rarely are PIOLs of T-cell lineage and if there is strong clinical evidence for suggesting such an entity (as in the setting of cutaneous T-cell lymphoma/mycosis fungoides) then we identify gene rearrangements in the T-cell receptor (TCR) gene by using primers for CDR3 and the variable region in the gamma chain (γ) gene [51].…”

“…Unfixed and unstained cytospin slides should be sent to a molecular lab, such as at the National Eye Institute (NEI), where microdissection of a minimal of 15 atypical cells may then be subjected to molecular analysis (Fig. 4) [24,25,50]. When using PCR to detect monoclonality [specifically, rearrangements of the immunoglobulin heavy chain (IgH) gene] of the malignant B-cell, the malignant cells most frequently involved in PIOL, knowledge of the typical primers used is essential.…”

“…It must be stressed that time is of the essence because lymphoma cells rapidly begin to degenerate. Centrifugation of the submitted sample at 1,000 rpm and pipeting the supernatant into another tube for enzymelinked immunosorbent assay (ELISA) assay and molecular investigation (PCR for infective virus) are recommended [23][24][25][29][30][31]. The sediment in the original tube is then resuspended in 0.2-1 ml of RPMI and cytocentrifuged again.…”

“…A standard three-port pars plana approach is employed. Because cytology [22] is standard and we frequently use molecular analysis with polymerase chain reaction amplification (PCR) [23][24][25] and cytokine-level analysis [26,27], a complete core vitrectomy is recommended. Undiluted vitreous is first obtained for cytological analysis.…”