Comparison of Reverse Transcription Quantitative Real-Time PCR, Flow Cytometry, and Immunohistochemistry for Detection of Monoclonality in Lymphomas

In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa？:？lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription quantitative real-time PCR analysis has complementary values. In a considerable number of cases tumor cells produced both kappa and lambda light chain transcripts, but only one type of light chain peptide was produced. 1. Introduction B lymphocytes produce immunoglobulins consisting of a heavy chain and either a kappa (IGKC) or a lambda (IGLC) light chain. Each B lymphocyte decides early by the rearrangement of its immunoglobulin genes which light chain to produce [1]. The excluded light chain gene is not properly rearranged or remains in germ line configuration [2]. In healthy humans 60–70% of the B cells produce kappa chains and the rest produce lambda chains [1, 3]. Normal lymphoid tissues therefore contain a mixture of B cells that express IGKC and IGLC at a ratio of about 60？:？40 = 1.5. Tumors of B cell origin are monoclonal and arise from one transformed cell. The single cell origin of malignant clones results in exclusive expression of IGKC or IGLC light chains in the vast majority of all B cell malignancies although B cell tumors that produce both kappa and lambda chains have been reported [4]. The clonal expression of IGKC or IGLC is thus used as an important diagnostic marker for B cell malignancies and currently determined on protein level by immunohistochemistry (IHC), flow cytometry (FC), or enzyme-linked immunosorbent assay techniques. Previously, we used reverse transcription quantitative real-time PCR (RT-qPCR) to quantify IGKC and IGLC gene transcripts in a small set of lymphomas and found that also gene expression level clonality was frequently evident [5]. In