Detection of malignant B lymphocytes by PCR clonality assay using direct lysis of cerebrospinal fluid and low volume specimens

Abstract

The diagnosis of lymphoid malignancies is often challenging in paucicellular specimens. PCR may also be limited by insufficient cells for DNA isolation and incomplete coverage of gene rearrangements. This study aims to evaluate a PCR method for IgH clonality using direct cell lysates. PCR amplification used cell lysate from detergent-based lysis and BIOMED-2 primers. CSF specimens were tested for 20 patients with primary CNS lymphoma or systemic lymphoma suspected for CNS involvement. Cytology and flow cytometry analysis was performed in parallel with PCR. Direct lysis produced a better yield than the column-based method for DNA isolation. PCR using lysate showed an efficiency of clonality detection from a minimum of 20 tumor cells. PCR clonality was found in nine of the 20 CSFs, and positive PCR was concordant with both cytology and flow cytometry in seven cases. There were two cases positive for PCR, but indeterminate for flow cytometry because of insufficient cell events. Of the eleven PCR-negative cases, two were considered as false negative, as flow cytometry showed positive for malignant cells. The PCR was also performed successfully with a specimen from the anterior chamber of the eye. PCR clonality with direct cell lysis of CSF is feasible, and it may overcome the limitation of DNA isolation. This PCR method may be particularly useful for small volume and low cell CSF when flow cytometry is inconclusive.