Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients.

BCR-ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR-ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR-ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR-ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR-ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR-ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR-ABL immunobead assay detects all types of BCR-ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within approximately 4 h, and can be run in parallel to routine immunophenotyping.

The relation of arterial stiffness to endothelial function in healthy subjects.

Local wall stiffness affects endothelial responsiveness but how global measures affect responsiveness is unanswered. We assessed this by comparing reactive hyperaemic responses of brachial diameter (RHRBD) with central (heart-to-brachial artery pulse wave velocity (PWV); large (C1)) and peripheral (C2) arterial stiffness. Twelve healthy subjects were investigated. RHRBD was induced via an upper- or forearm occluding cuff. Arterial diameter changes were measured using echo ultrasound. Arterial stiffness and RHRBD were compared using a Pearson correlation coefficient (r) and Bland-Altman analysis of Z-scores (indicated as 95% confidence intervals (CI) and expressed in units of standard deviation (SD) from the mean). Weak relations were found between upper-arm RHRBD responses and C2 (r = 0.56, P = 0.06; 95% CI +/- 1.84 SDs) and C1 (r = 0.55, P = 0.06; 95% CI +/- 1.86 SDs). An inverse relation was found between upper-arm RHRBD responses and PWV (r = -0.55, P = 0.06), but Bland-Altman plots revealed no agreement between these parameters (P > 0.05; 95% CI +/- 3.46 SDs). Forearm RHRBD were not related to PWV, C1 or C2 (P > 0.05; 95% CI > 2 SDs). The weak relation between upper-arm endothelial responses and C2 and C1 seems to suggest that C2, and also C1, is not a good and reliable method for assessments of endothelial health. Furthermore, if anything, upper-arm mediated RHRBD responses are more affected by arterial stiffness than forearm responses.