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Chronic lymphocytic leukaemia (CLL) follows a variable clinical course with patient survival ranging from only a few years despite treatment, to several decades in patients who may never require clinical intervention. Determination of the mutational status of a patient's immunoglobulin heavy chain variable region (Ig V(H)) gene has been used to provide prognostic information, but this assay is not available in most laboratories. The discovery of the expression of the protein tyrosine kinase zeta-associated protein (ZAP)-70 in V(H)-unmutated CLL cases led to its proposal as a surrogate marker for V(H) status. This study investigated the measurement of ZAP-70 expression in CLL using different flow cytometric protocols. Two different antibodies and two different staining methods were compared. The Caltag ZAP-70 antibody and Fix & Perm kit were the easiest to use and were the most sensitive and specific combination, with 91% concordance between ZAP-70 expression and V(H) status. Three patients (9%) were discordant (two V(H) mutated/ZAP-70 positive, and one V(H) unmutated/ZAP-70 negative). No correlation existed between CD38 and either ZAP-70 expression or V(H) status. Measurement of ZAP-70 expression using the Caltag antibody/kit combination provides a standardized flow cytometric method that could be introduced into a routine CLL immunophenotyping panel in a clinical diagnostic laboratory.

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Summary We have used the polymerase chain reaction to detect DNA sequences related to human papillomavirus type 16, by simultaneous priming with oligonucleotides from the E6 and LI/L2 open reading frames of the HPV16 genome. The HPV16-related sequence is present at low levels in normal oral tissue, in addition to biopsies and cell cultures from patients with benign and malignant disease. Ultimate analysis of the amplified sequences from the E6(120bp) and Ll/L2(173bp) regions of HPV16 was achieved by gel electrophoresis and comparative nucleotide sequencing. The oral carcinoma biopsies and tissue cultures contained DNA sequences which were identical to the E6 region of HPV16, but only rarely contained sequences closely related to the L1/L2 region. The PCR technology should permit the detection, identification and cloning of latent viruses from extremely small tissue biopsies.