Kinetic titration method in flow cytometry for quantification of cell receptors.

For the quantitative determination of cell receptors by fluorescence flow cytometry, we proposed a new method, which takes into account the reaction kinetics. The binding reaction of the ligand with receptors begins after placing the cells in the ligand solution. In the proposed method, there are several samples with the same concentration of cells and different initial concentrations of fluorescently labeled ligand, and each sample is measured by a flow cytometer once at the time when the following condition is met: the product of the incubation time (cells with ligand) and the initial concentration of ligand is the same for all samples. The proposed approach eliminates disadvantages and combines advantages of both kinetic and titration methods for quantification of receptors on single cells without the use of traditional calibration fluorescent beads. Practical application of the method was demonstrated in quantification of CD8 and CD14 on peripheral blood human leukocytes. Particularly, we found decreased (by a factor of two) mean number of CD14 on monocytes and granulocytes in patients with atherosclerosis (treated in the hospital) compared to conditionally healthy donors, whereas no difference was found in the mean CD8 expression on leukocytes between the same patient and donor groups.

Blood platelet quantification by light scattering: from morphology to activation.

Analysis of blood platelets encounters a number of different preanalytical issues, which greatly decrease the reliability and accuracy of routine clinical analysis. Modern hematology analyzers determine only four parameters relating to platelets. Platelet shape and dose-dependent activation parameters are outside the scope of commercial instruments. We used the original scanning flow cytometer for measurement of angle-resolved light scattering and the discrete dipole approximation for simulation of light scattering from a platelet optical model, as an oblate spheroid, and global optimization with two algorithms: the DATABASE algorithm to retrieve platelet characteristics from light scattering and the DIRECT algorithm to retrieve dose-dependent activation parameters. We developed the original sampling protocol to decrease spontaneous platelet activation. The new protocol allows us to keep most of the platelets in resting and partially activated states before analysis. The analysis delivers 13 content and morphological parameters of the platelets. To analyze platelet shape change during ADP activation we developed a phenomenological model. This model was applied to the analysis of ADP activation of platelets to give 8 dose-dependent activation parameters. To demonstrate the applicability of the developed protocol and analytical method, we analyzed platelets from five donors. This novel approach to the analysis of platelets allows the determination of 21 parameters relating to their content, morphology and dose-dependent activation.

Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes.

We propose a calibration-free method to determine the number of receptors per cell, as well as the direct and the reverse reaction rate constants for a single receptor. The method is based on the analysis of the temporal evolution of the cells mean fluorescent intensity measured by a flow cytometer during the ligand-receptor (antigen-antibody) binding under the conditions of their comparable concentrations. We developed the kinetic approach accounting both for the delay between the dilution and the measurement and for the practical duration of the measurement itself. The method was applied to determine thenumber of CD14 receptors on human blood mononuclear (granulocytes, monocytes, lymphocytes) cells of several donors. We also obtained the direct ( k+= (5.6 ± 0.2) × 107 M-1 min-1 ) and reverse ( k-= (1.3 ± 0.2) × 10-2 min-1 ) rate constants of ligand-receptor interaction, and estimated the size of the binding site as b = 0.5 nm. The latter allows one to recalculate the rate constants for a different ligand, fluorescent label, medium viscosity, and/or temperature. The knowledge of the rate constants is essential for the calibration-free determination of the number of receptors per cell from a single kinetic curve of the cells mean fluorescence intensity.

Randomized trial of fish oil infusion to prevent atrial fibrillation after cardiac surgery: data from an implantable continuous cardiac monitor.

OBJECTIVE: To test the hypothesis that perioperative OMEGA-3 polyunsaturated fatty acid infusion would reduce the incidence of postoperative atrial fibrillation in patients after coronary artery bypass grafting as assessed by an implantable continuous cardiac monitor. DESIGN: Prospective, randomized, double-blind, placebo-controlled study. SETTING: Tertiary cardiothoracic referral center. PARTICIPANTS: Thirty-nine patients with coronary artery disease who underwent surgery with cardiopulmonary bypass. INTERVENTIONS: Patients were assigned randomly to receive either OMEGA-3 polyunsaturated fatty acids (200 mg/kg/day starting before anesthesia induction for 24 hours followed by 100 mg/kg/day for 7 days) or placebo. The primary outcome was freedom from atrial fibrillation at 2-year follow-up. Reveal(®) cardiac monitor was implanted subcutaneously in all patients. Data from the cardiac monitor were collected on the 10th day and 3, 6, 12, and 24 months postoperatively. MEASUREMENT AND MAIN RESULTS: Postoperative atrial fibrillation developed in 4 (19%) patients in the control group and in 5 (27.8%) patients in the study group at 10-day follow-up (p = 0.88). At 2-year follow-up, 5 (27.8%) patients in the control group and 6 (35.3%) patients in the study group had atrial fibrillation (p = 0.9). Atrial fibrillation duration predicted risk of cardiovascular hospitalization at the 2-year follow-up (regression coefficient estimate = 0.24, standard error 0.02, p<0.0001; R(2) = 0.74). CONCLUSIONS: Infusion of OMEGA-3 polyunsaturated fatty acids failed to prevent the occurrence of atrial fibrillation in 2 years after coronary artery bypass grafting surgery. The cumulative atrial fibrillation duration registered by the continuous cardiac monitor at the 2-year follow-up was a significant predictor of an adverse outcome.

Infusion of 7.2% NaCl/6% hydroxyethyl starch 200/0.5 in on-pump coronary artery bypass surgery patients: a randomized, single-blind pilot study.

BACKGROUND: NaCl 7.2%/6% hydroxyethyl starch (HES) 200/0.5 (HSH) has shown its beneficial effects in cardiac surgery and immunomodulatory values in experiment and human studies. However, there is concern regarding detrimental renal effects of chloride and HES in the intensive care setting. OBJECTIVES: The objective of this study was to evaluate the influence of HSH on kidney integrity and the level of inflammatory mediators in on-pump coronary artery bypass surgery patients. DESIGN: This was a single-center, prospective, randomized, single-blind pilot study. SETTING: This work was performed at a tertiary cardiothoracic referral center during the period from February to August 2012. PATIENTS: Forty patients scheduled for on-pump coronary artery bypass surgery were included. INTERVENTIONS: PATIENTS were randomized to receive once either 7.2% NaCl/6% HES 200/0.5 (HSH group, n = 20) or placebo (0.9% NaCl; control group, n = 20) at a dose of 4 mL·kg for 30 min after anesthesia induction. MAIN OUTCOME MEASURES: The primary end point was the incidence of acute kidney injury (AKI) defined according to the Kidney Disease: Improving Global Outcomes. Secondary end points were peak serum cystatin C, peak urine neutrophil gelatinase-associated lipocalin, plasma levels of interleukin 6 (IL-6), IL-10, intercellular adhesion molecule 1, and endothelial-leukocyte adhesion molecule (E-selectin). RESULTS: The incidence of AKI within 48 h was similar between the groups (HSH: four patients [20%]; control: six patients [30%]). There was a significantly lower peak value for serum cystatin C in the HSH group (0.83 [0.73-0.89] mg·L) compared with the control group (1.02 [0.88-1.15] mg·L; P = 0.001). PATIENTS in both groups had similar peak postoperative urine neutrophil gelatinase-associated lipocalin concentrations. NaCl 7.2%/6% hydroxyethyl starch 200/0.5 significantly reduces levels of IL-6 and IL-10 at 4 h after cardiopulmonary bypass and intercellular adhesion molecule 1 and E-selectin at 4 h after cardiopulmonary bypass and on postoperative day 1 (P < 0.05 for all). CONCLUSIONS: NaCl 7.2%/6% hydroxyethyl starch 200/0.5 does not lead to the increase in AKI incidence when used for the volume therapy in on-pump coronary artery bypass surgery patients. NaCl 7.2%/6% hydroxyethyl starch 200/0.5 usage enhanced neither tubular injury nor alteration of glomerular filtration. In addition, HSH can reduce the level of the inflammatory mediators after surgery.

Hypothermic versus normothermic cardiopulmonary bypass in patients with valvular heart disease.

OBJECTIVE: The aim of this study was to test the hypothesis that normothermic cardiopulmonary bypass (CPB) is as effective as hypothermic CPB in terms of cardiac protection (cTnI level) and outcome in patients with valvular heart disease. DESIGN: Prospective randomized study. SETTING: A tertiary cardiothoracic referral center. PARTICIPANTS: 140 patients who had valvular heart disease, with/without coronary artery disease, surgically treated under CPB. INTERVENTIONS: The patients were allocated randomly to undergo either hypothermic (temperature [T], 31 °C-32 °C) or normothermic CPB (T>36 °C). MEASUREMENTS AND MAIN RESULTS: The primary endpoint was the dynamics of troponin I. The secondary endpoints were ventilation time, the need for inotropic support, intensive care unit (ICU) and hospital stay durations, complications, and mortality. There were no significant intergroup differences in dynamics of troponin I. Ventilation time was significantly lower in the hypothermic group (6 (5-9) and 8 (5-12); p = 0.01). CONCLUSIONS: Normothermic CPB in patients with valvular heart disease was as effective as hypothermic perfusion in terms of myocardial protection after the surgery assessed by cTnI release. The short ventilation duration in patients who underwent hypothermic CPB needs to be confirmed in a future investigation.

Evaluation of nutritional screening tools among patients scheduled for heart valve surgery.

BACKGROUND AND AIM OF THE STUDY: The study aim was to detect the most sensitive nutritional screening tool and to assess its prognostic value with regards to an adverse clinical course in patients with heart valve disease undergoing cardiopulmonary bypass (CPB). METHODS: This prospective cohort study included 441 adult patients who were screened using four nutritional screening tools: Nutritional Risk Screening 2002 (NRS-2002); Malnutrition Universal Screening Tool (MUST); Mini Nutritional Assessment (MNA); and Short Nutritional Assessment Questionnaire (SNAQ). Nutritional assessment was performed using a Subjective Global Assessment (SGA). In-hospital mortality, postoperative complications, and duration of hospital stay were each analyzed. RESULTS: With regards to the detection of malnutrition, the sensitivities of MUST, SNAQ, MNA and NRS-2002 were 100%, 92%, 84.6% and 43.6%, respectively. Malnutrition identified by MUST and MNA were associated with postoperative complications (OR 1.63, p = 0.033 and OR 1.6, p = 0.035) and prolonged hospitalization (OR 1.57, p = 0.048 and OR 1.7, p = 0.02). According to multivariate logistic regression analysis, along with well-known age and duration of CPB, malnutrition identified by MUST and MNA was associated with a risk of development of complications (OR 1.6, p = 0.049 and OR 1.6, p = 0.04, respectively). The sensitivities of SNAQ, MUST, NRS-2002 and MNA with regards to postoperative complications were 26.8%, 28.8%, 10%, and 31.6%, respectively. CONCLUSION: The MUST tool is preferable with regards to the detection of malnutrition. Both, MUST and MNA independently predicted postoperative complications. SNAQ and NRS-2002 proved insensitive with regards to the postoperative course among patients with heart valve disease who were scheduled for cardiothoracic surgery.

Hypertonic solution decreases extravascular lung water in cardiac patients undergoing cardiopulmonary bypass surgery.

OBJECTIVE: To test the hypothesis that the infusion of hypertonic solution would decrease extravascular lung water postoperatively and thus improve pulmonary function. DESIGN: Prospective, randomized, blinded trial. SETTING: Tertiary cardiothoracic referral center. PARTICIPANTS: Twenty-six patients with coronary artery disease who underwent surgery with cardiopulmonary bypass (CPB). INTERVENTIONS: Patients were allocated randomly to receive 4 mL/kg of 7.2% NaCl/hydroxyethyl starch, 200/0.5 (HSH group) or an equal volume of 0.9% NaCl (control group) for 30 minutes starting after anesthesia induction. The extravascular lung water index, hemodynamic and biochemical data, and the rate of complications were analyzed. MEASUREMENTS AND MAIN RESULTS: The extravascular lung water index was significantly lower (7 v 9.5 mL/kg) in the HSH group at the first postoperative day (p < 0.01). The index of arterial oxygenation efficiency was significantly higher at 5 minutes and 2 and 4 hours after cardiopulmonary bypass (CPB) in the HSH group (p < 0.05). The alveolar-arterial oxygen tension difference was significantly lower at 5 minutes and 2 and 4 hours after CPB in the HSH group (p < 0.01). The cardiac index was significantly higher at 5 minutes after infusion in the HSH group (p < 0.05). CONCLUSIONS: The infusion of HSH leads to significant decreases in the extravascular lung water index during and after cardiac surgery and is associated with better preservation of pulmonary function and transient increases in the cardiac index. Further trials are needed to clarify the clinical advantages of hypertonic solution administration in patients undergoing surgery with CPB.