Isolated evidence of clonality in biopsy material is therefore an insufficient diagnostic criterion to determine malignancy. the same run. PCR analysis of and IgH gene rearrangements DNA was isolated from paraffin wax embedded biopsy material as described previously . PCR analysis was adapted from McCarthy PCR analysis was performed using VBJB2 and DB1JB2 primer combinations . TCRB and TCRG PCR products were electrophoresed through a 10% polyacrylamide gel. These TCR PCR methods were established using DNA from 16 clonal T cell tumours, characterized previously by Southern blot analysis, as clonal for PD168393 and loci. In these experiments clonal gene rearrangements and VJ and DJ gene rearrangements were detected in 94% and 71% of cases, respectively (data not shown). IgH FR2 PCR analysis was performed using a seminested PCR amplification method . IgH FR3 PCR analysis was performed using FR3 and JH primers . PCR products were electrophoresed through a 5% (FR2) or 10% (FR3) polyacrylamide gel. Our detection rate is 88% for demonstrating B cell clonality in a series of 40 immunohistologically characterized B cell neoplasms including 24 B cell leukaemias (22 B cell chronic lymphocytic leukaemia, one B cell prolymphocytic leukaemia and one hairy cell leukaemia) and 16 B cell non-Hodgkin’s lymphoma (data not shown). For all analyses, clonal bands are defined as one or two narrow sharp intense bands visible on polyacrylamide gels after electrophoresis, whereas polyclonal PCR products appear as a smear within the appropriate size range. Oligoclonal populations are defined by three or more distinct bands. Sensitivity DNA from known positive clonal controls were diluted serially (comprising 100, 50, 40, 30, 20, 10, 5, 2 and 1% of tumour DNA) with DNA from reactive polyclonal controls. Following appropriate PCR amplification, clonal products could be detected at a level of 2% clonal DNA on a polyclonal background smear for all PCR protocols. Statistics Data were analysed using MannCWhitney 005. RESULTS We were able to assess tissue from 34 patients (18 female and 16 male), of whom PD168393 19 patients had progressed to lymphoma (follow up 12C160 years) and 15 had not (follow up 71C304 years). Of those patients with a lymphoma, 16 (seven female, nine male) had a diagnosis of CVID, two subclass deficiency (female) and one XLA (male). Of those patients without lymphoma, 13 (eight female, five male) had a diagnosis of CVID, one of IgG subclass deficiency (female) and one X-linked immunoglobulin deficiency of uncertain aetiology (male). Figure 1 shows the age and gender of patients at time of diagnosis of lymphoma. The median age of diagnosis of lymphoma was 45 years (range 20C69); for females it was 46 years (range 20C50, = 9) and males 42 years (range PD168393 26C69, = 10) (not significant, = 072). The median age at time of suspected lymphoma (as indicated by time of first PD168393 biopsy) and diagnosis of CVID or subclass deficiency was Rabbit Polyclonal to PTPRZ1 comparable in women with and without lymphoma (Table 1). In men there was a significant difference between the age at first biopsy in those with and without lymphoma (= 003 for all data, = 004 if XLA excluded), but not at the age of diagnosis of antibody deficiency. This is likely to relate to a skewed distribution of ages as shown by the minimum age for antibody deficiency (6 24 years, see Table 1). Open in a separate window Fig. 1 Age and gender at diagnosis of lymphoma. Data (*) include all patients presenting with lymphoma during the study period, including one patient in whom the histology was indicative of lymphoma, but equivocal (see text). Table 1 Analysis of age at diagnosis of antibody deficiency (XLA excluded), gender and lymphoma occurrence hybridization in 17 biopsies and was not detected in either the lymphoma biopsies (10/17).