Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a disorder in which the tempo of disease progression is highly variable, and prognostic markers that can be utilized at diagnosis are regarded as clinically important. Currently, there are several prognostic factors, such as immunoglobulin heavy chain (IgVH) mutational status, and ZAP-70 protein expression in neoplastic B-cells, that have demonstrated significant discriminative power in the prognostication of CLL. They are, however, largely unavailable in the routine diagnostic laboratory setting. METHODS: In this study, we characterized the IgVH status and ZAP-70 expression by molecular techniques in a cohort of 108 patients with CLL, and correlated these results with three different methods of ZAP-70 expression by flow cytometry. We then assessed the results of these methods in terms of prognostic power as characterized by time to first treatment (TTFT). RESULTS: By comparing three different flow cytometry methods using receiver­operator curve (ROC) analysis, we identified that by utilizing a corrected mean fluorescence intensity (CorrMFI) algorithm for assessing ZAP-70 expression, there was good correlation with both IgVH mutational status, and ZAP-70 expression as assessed by qPCR. We were also able to show that ZAP-70 expression, as assessed by both qPCR and the CorrMFI method, was prognostic of TTFT. CONCLUSIONS: While confirmation in a larger patient cohort, with longer follow-up is required, we believe that the CorrMFI represents the most promising method currently available in a routine diagnostic setting for the assessment of ZAP-70 expression in CLL patients.

Using HitAlert flow cytometry to detect heparin-induced thrombocytopenia antibodies in a tertiary care hospital.

We aimed to assess the utility of HitAlert flow cytometry as a diagnostic functional heparin-induced thrombocytopenia (HIT) assay in a tertiary care hospital. The 4Ts score was used to assess pretest probability of HIT in 37 patients. Serum was analysed for HIT antibodies by the flow cytometry HitAlert assay. Results were compared with an antigenic assay, the particle gel immunoassay, PaGIA ID PF4/Hep Ab assay; and two functional assays, the Multiplate whole blood impedance aggregometry assay (WBIA), and the serotonin release assay (SRA). Flow cytometry was positive in 14 out of 37 patients, including zero out of eight, five out of 19 and nine out of 10 in the low, intermediate and high-risk groups by 4Ts score, respectively. Using the SRA as a 'gold standard', flow cytometry has a sensitivity of 81% and a specificity of 100% for the diagnosis of HIT. The other functional assay (WBIA) had similar sensitivity (81%) and specificity (90%) to flow cytometry. In contrast, the PaGIA maintained a high sensitivity of 100% but a specificity of only 20%. The improved specificity of flow cytometry over the antigenic assay was most marked in the 4T intermediate-risk group in which similar results were obtained between all three functional assays. We demonstrate that compared with an immunological assay (PaGIA), flow cytometry can improve the specificity of laboratory diagnosis of HIT without loss of sensitivity using SRA as a standard. Flow cytometry may have a role in the first-line laboratory diagnosis of HIT, especially when combined with an immunological assay such as PaGIA.