The use of CellaVision™ DM96 in the verification of the presence of blasts in samples flagged by the Sysmex XE-5000.

INTRODUCTION: The CellaVision™DM96 is a digital pattern recognition system that classifies white blood cells. The aim of this study was to evaluate whether the CellaVision preclassification feature, without a subsequent re-classification, was a sufficient approach to follow up flags reported by Sysmex XE-5000. METHODS: Pairs of blood smears from 400 samples reported with suspect flags were examined using conventional microscopy and the CellaVision features. The effect of pre- vs. re-classification, and intersmear and between-technologist variation, on blast counts was assessed using generalized linear mixed models (GLMM). RESULTS: The GLMM analysis showed a significant difference between the blast counts of preclassification vs. re-classification (P = 0.009). The analysis showed no significant difference between duplicate smears (P = 0.621) or between technologists (P = 0.542). Preclassification showed blasts in 105 samples (26%), where the re-classification feature did not detect any blasts. Not a single sample that was re-classified as positive for blasts was preclassified as negative. Compared to manual microscopy, the sensitivity and specificity of the preclassification feature were 0.83 and 0.66, respectively. CONCLUSION: The preclassification feature alone is sufficient to verify the absence of blasts in flagged samples. When the preclassification feature detects blasts, the finding has to be verified or reclassified by an experienced technologist. However, the use of CellaVision™DM96 in the follow-up of blast reports has to be questioned due to the finding of false-negative samples in the preclassification feature, but also after re-classification, compared to manual slide review.

Anticoagulant effects of an antidiabetic drug on monocytes in vitro.

INTRODUCTION: Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties. METHODS: We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RT-qPCR, western blot) and on cell viability and MP release (flow cytometry). RESULTS: Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs. CONCLUSIONS: Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated.

Calcium ionophore-induced de-encryption of tissue factor in monocytes is associated with extensive cell death.

INTRODUCTION: Cell surface tissue factor (TF) is normally encrypted, but can be activated by various cellular perturbations. Exposure of TF bearing cells to calcium ionophore has been reported to increase TF activity, de-encrypt TF, by phosphatidylserine (PS)-dependent and -independent mechanisms. Our aim has been to examine at the single cell level, if increased cell surface PS coincided with increased cell surface TF antigen, and cell death (necrosis, 7-AAD-intercalation), and relate this to monocyte- and microparticle (MP)-associated procoagulant activity. MATERIALS AND METHODS: We exposed lipopolysaccharide-stimulated, human, elutriation-purified, cryopreserved TF bearing monocytes to increasing concentrations of calcium ionophore (A23187) and measured procoagulant activity in cells and supernatants. These measurements were compared with quantification of cell surface TF and PS (Annexin V) and of cell necrosis (7-AAD) by flow cytometry, and complemented by confocal microscopy. RESULTS: We observed that calcium ionophore increased cellular and MP-associated TF activity, but not cell surface TF antigen. The discrepancy between TF activity and TF antigen coincided with a dose-dependent increase in the number of cells expressing PS. These cells were to a large extent necrotic and many of them also expressed TF. CONCLUSIONS: We suggest such TF positive dying cells to contribute to the discordance between TF activity and TF expression. Calcium ionophore also increased MP-associated TF activity and release of MPs may be a way to disseminate procoagulant activity. Our findings emphasize the importance of adequately assessing cell death and taking into consideration its possible role in experiments with calcium ionophore.