Flow cytometric assessment of agonist-induced P-selectin expression as a measure of platelet quality in stored platelet concentrates.

BACKGROUND: Platelet (PLT) function in PLT concentrates declines during storage and is further affected by pathogen reduction treatment. Flow cytometric assessment of agonist-induced P-selectin expression can be used to assess PLT function in patients with thrombocytopenia. The aim of this study was to evaluate how this functional test relates to established in vitro measures of PLT function. STUDY DESIGN AND METHODS: Six units of PLTs in plasma and 6 units of riboflavin and ultraviolet (Mirasol, TerumoBCT)-treated PLTs in plasma were sampled on Days 2, 6, 8, and 10 after donation. PLT concentration, Annexin 5A staining, ThromboLUX (LightIntegra) thrombelastography, and P-selectin expression, both in unstimulated PLTs and in response to concentration series of adenosine diphosphate, collagen-related peptide, and thrombin receptor-activating peptide (TRAP), were measured. RESULTS: For PLTs in plasma Annexin 5A expression increased by 0.60% (95% confidence interval [CI], 0.40%-0.80%) and P-selectin expression increased by 1.2% (95% CI, 0.80%-1.6%) per day. Responsiveness to TRAP simultaneously decreased by 1.3% (95% CI, 0.80%-1.8%) per day. After Mirasol treatment ThromboLUX scores decreased 3.3 points (95% CI, 0.2-6.4 points) from 22 to 19 points, Annexin 5A expression increased by 4.8% (95% CI, 3.3%-6.2%), and P-selectin expression increased by 13% (95% CI, 10%-16%), all averaged over the entire storage period. Responsiveness to TRAP simultaneously decreased by 19% (95% CI, 17%-21%). CONCLUSIONS: Our results suggest flow cytometric measurement of agonist-induced P-selectin expression can measure PLT quality decline over the entire range encountered during 10-day storage of both standard PLTs and Mirasol-treated PLTs in plasma.

CD34+CD38- leukemic stem cell frequency to predict outcome in acute myeloid leukemia.

Current risk algorithms are primarily based on pre-treatment factors and imperfectly predict outcome in acute myeloid leukemia (AML). We introduce and validate a post-treatment approach of leukemic stem cell (LSC) assessment for prediction of outcome. LSC containing CD34+CD38- fractions were measured using flow cytometry in an add-on study of the HOVON102/SAKK trial. Predefined cut-off levels were prospectively evaluated to assess CD34+CD38-LSC levels at diagnosis (n = 594), and, to identify LSClow/LSChigh (n = 302) and MRDlow/MRDhigh patients (n = 305) in bone marrow in morphological complete remission (CR). In 242 CR patients combined MRD and LSC results were available. At diagnosis the CD34+CD38- LSC frequency independently predicts overall survival (OS). After achieving CR, combining LSC and MRD showed reduced survival in MRDhigh/LSChigh patients (hazard ratio [HR] 3.62 for OS and 5.89 for cumulative incidence of relapse [CIR]) compared to MRDlow/LSChigh, MRDhigh/LSClow, and especially MRDlow/LSClow patients. Moreover, in the NPM1mutant positive sub-group, prognostic value of golden standard NPM1-MRD by qPCR can be improved by addition of flow cytometric approaches. This is the first prospective study demonstrating that LSC strongly improves prognostic impact of MRD detection, identifying a patient subgroup with an almost 100% treatment failure probability, warranting consideration of LSC measurement incorporation in future AML risk schemes.

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Platelet function in adult ITP patients can be either increased or decreased, compared to healthy controls, and is associated with bleeding risk.

OBJECTIVES: To test whether, together with platelet count, platelet activity could be an important predictor of bleeding risk in immune thrombocytopenia (ITP) patients. METHODS: Platelet activity was tested by flow cytometric measurement of agonist induced P-selectin expression and compared between 23 adult ITP patients and 22 healthy volunteers. RESULTS: Platelet activity could be either increased or decreased in ITP patients, compared to healthy volunteers. In the lowest platelet count category, normal to low platelet activity was associated with the biggest increase in bleeding risk. Risk difference 80% (95% confidence interval: 45-115%) for <32 × 10(9) platelets/L. For higher platelet counts, there was no association of platelet activity with bleeding risk. DISCUSSION: Increased platelet activity was associated with decreased bleeding risk, but only in patients with low platelet counts. CONCLUSION: Platelet activity can be a predictor of bleeding risk in ITP patients with low platelet counts.

The relationship between impulsive choice and impulsive action: a cross-species translational study.

Maladaptive impulsivity is a core symptom in various psychiatric disorders. However, there is only limited evidence available on whether different measures of impulsivity represent largely unrelated aspects or a unitary construct. In a cross-species translational study, thirty rats were trained in impulsive choice (delayed reward task) and impulsive action (five-choice serial reaction time task) paradigms. The correlation between those measures was assessed during baseline performance and after pharmacological manipulations with the psychostimulant amphetamine and the norepinephrine reuptake inhibitor atomoxetine. In parallel, to validate the animal data, 101 human subjects performed analogous measures of impulsive choice (delay discounting task, DDT) and impulsive action (immediate and delayed memory task, IMT/DMT). Moreover, all subjects completed the Stop Signal Task (SST, as an additional measure of impulsive action) and filled out the Barratt impulsiveness scale (BIS-11). Correlations between DDT and IMT/DMT were determined and a principal component analysis was performed on all human measures of impulsivity. In both rats and humans measures of impulsive choice and impulsive action did not correlate. In rats the within-subject pharmacological effects of amphetamine and atomoxetine did not correlate between tasks, suggesting distinct underlying neural correlates. Furthermore, in humans, principal component analysis identified three independent factors: (1) self-reported impulsivity (BIS-11); (2) impulsive action (IMT/DMT and SST); (3) impulsive choice (DDT). This is the first study directly comparing aspects of impulsivity using a cross-species translational approach. The present data reveal the non-unitary nature of impulsivity on a behavioral and pharmacological level. Collectively, this warrants a stronger focus on the relative contribution of distinct forms of impulsivity in psychopathology.