Single-cell physical phenotyping of mechanically dissociated tissue biopsies for fast diagnostic assessment

Rapid and accurate histopathological diagnosis during surgery is critical for clinical decision-making. The prevalent method of intraoperative consultation pathology is time, labour and cost intensive and requires the expertise of trained pathologists. Here, we present an alternative technique for the rapid, label-free analysis of biopsy samples by sequentially assessing the physical phenotype of singularized, suspended cells in high-throughput. This new diagnostic pipeline combines enzyme-free, mechanical dissociation of tissues with real-time deformability cytometry at measurement rates of 100 – 1,000 cells/sec, and machine learning-based analysis. We show that physical phenotype parameters extracted from brightfield images of single cells can be used to distinguish subpopulations of cells in various tissues, without prior knowledge or the need for molecular markers. Further, we demonstrate the potential of our method for inflammatory bowel disease diagnostics. Using unsupervised dimensionality reduction and logistic regression, we accurately differentiate between healthy and tumorous tissue in both mouse and human biopsy samples. The method delivers results within 30 minutes, laying the groundwork for a fast and marker-free diagnostic pipeline to detect pathological changes in solid biopsies.

Abstract 240: Comparison of Hemodynamics Generated by Synchronized and Interposed Compressions During Treatment of Pseudo Electro-mechanical Dissociation

Introduction: Pseudo electro-mechanical dissociation (P-EMD) is a cardiac arrest variant characterized by a life-threatening reduction in cardiac output in the presence of organized electrical activity. Synchronization of chest compressions to the R-wave in the ECG may be preferable to the delivery of standard CPR. However, in the bradycardic P-EMD state, synchronization may result in inadequate blood flow due to the low compression/heart rate. This pilot study examined the hemodynamic effect of interposing additional chest compressions between synchronized chest compressions during bradycardic P-EMD to increase the compression rate. Methods: P-EMD was induced via hypoxia in three female swine (~30 kg) and treated with synchronized compressions until the onset of asystole (HR<12 BPM). Interposed compressions were added when the heart rate fell below 60 BPM. A chest compression was classified as synchronized or interposed depending on the presence or absence of a co-incident R-wave. Hemodynamic parameters were integrated or averaged over each compression interval. Results: Synchronized compressions tended to produce larger aortic pressures, larger carotid blood flows, and lower right atrial pressures than interposed compressions. Data from one experiment are shown in Figure 1. The relative hemodynamic benefit of a synchronized chest compression appears to depend on the effectiveness of the underlying heart contraction. The interposed chest compressions generated forward carotid blood flow and increased the compression rate during bradycardia. Discussion: During bradycardic P-EMD, synchronized compressions may generate better hemodynamics than interposed compressions, and the combination of synchronized and interposed compressions may result in more blood flow than the delivery of synchronized compressions alone. Figure 1. Comparison of hemodynamics generated by synchronized compressions (blue) and interposed compressions (red).

Isolation of Skeletal Muscle-derived Cells Modeling Neural Crest-Derived Stem Cells for Therapeutic Use in Regenerative Periodontology

Periodontitis is microbial infection affecting periodontium, the tooth supporting structure and affects &gt;743 million people worldwide. Neural crest-derived stem cells (NCSCs) hold the promise to regenerate the damaged periodontium. These cells have been identified within adult adipose tissue, periodontal ligament, and palatal tissue. Typical enzymatic isolation protocols are expensive, time consuming and often not clinically compliant. Enzyme-free, mechanical dissociation has been suggested as an alternative method of generating cell suspensions required for cell separation and subsequent expansion ex vivo. In our study, samples of rat skeletal muscle tissue were used to appraise the suitability of a novel mincing method of mechanical dissociation against enzymatic digestion with collagenase and dispase. Skeletal muscle is readily available and has been shown to contain NCSC populations. We used a Rigenera-Human Brain Wave&reg; prototype mincer to produce a suspension of skeletal muscle-derived cells modeling NCSCs. We have compared the resulting cell cultures produced via mechanical dissociation and enzymatic dissociation, producing single cell suspensions suitable for Magnetic Cell Sorting (MACs) and Fluorescence-activated cell sorting (FACS). Despite the Countess Automated Cytometry data demonstrating that cell suspensions produced by mechanical dissociation (n=24) contain on average 26.8 times as many viable cells as enzymatic cell suspensions (n=18), enzymatic suspensions produced more successful cell cultures. Spheroids and subsequently adherent cells formed from 4 enzymatic cell suspensions (44.4%) vs. 1 mechanical cell suspension (8.3%). Enzymatic digestion protocols formed spheroids faster and more plentifully than mechanical cell suspensions. Adherent cells and spheroids isolated via both methods appear morphologically similarly to NCSCs from our previous studies.

Isolation of Neural Crest-derived Stem Cells for Therapeutic Use in Regenerative Periodontology

Periodontitis is microbial infection affecting periodontium, the tooth supporting structure and affects &gt;743 million people worldwide. Neural crest-derived stem cells (NCSCs) hold the promise to regenerate the damaged periodontium. These cells have been identified within adult adipose tissue, periodontal ligament, and palatal tissue. Typical enzymatic isolation protocols are expensive, time consuming and often not clinically compliant. Enzyme-free, mechanical dissociation has been suggested as an alternative method of generating cell suspensions required for cell separation and subsequent expansion ex vivo. In our study, samples of rat skeletal muscle tissue were used to appraise the suitability of a novel mincing method of mechanical dissociation against enzymatic digestion with collagenase and dispase. Skeletal muscle is readily available and has been shown to contain NCSC populations. We used a Rigenera-Human Brain Wave&reg; prototype mincer to produce a suspension of NCSCs. We have compared the resulting cell cultures produced via mechanical dissociation and enzymatic dissociation, producing single cell suspensions suitable for Magnetic Cell Sorting (MACs) and Fluorescence-activated cell sorting (FACS). Despite the Countess Automated Cytometry data demonstrating that cell suspensions produced by mechanical dissociation (n=24) contain on average 26.8 times as many viable cells as enzymatic cell suspensions (n=18), enzymatic suspensions produced more successful cell cultures. Spheroids and subsequently adherent cells formed from 4 enzymatic cell suspensions (44.4%) vs. 1 mechanical cell suspension (8.3%). Enzymatic digestion protocols formed spheroids faster and more plentifully than mechanical cell suspensions. Adherent cells and spheroids isolated via both methods appear morphologically similarly to NCSCs from our previous studies.

Abstract 439: Using the Time Interval Between R-Wave Detection and Maximum Compression Pressure to Target and Validate Delivery of Synchronized Chest Compressions During Pseudo Electro-Mechanical Dissociation

Introduction: The treatment of pseudo electro-mechanical dissociation (P-EMD) with standard chest compressions leads to some compressions that interfere with blood flow created by ventricular contraction and others that are synergistic. We have previously reported that the hemodynamics generated by standard chest compressions (StdCPR) depended on the time interval between the R-wave and the maximum compression pressure (t_int). Our goal was to use the t_int to identify the optimal timing for compression synchronization and to validate the delivery of synchronized chest compressions. Methods: Eight animals underwent surgical preparation and were exposed to hypoxia to induce P-EMD. The treatment period was divided into eight 45 sec epochs during which the P-EMD was left untreated or was treated with StdCPR or chest compressions synchronized to the R-wave in the ECG (SyncCPR). For each heart beat t_int was calculated as t peak AOP - t Rwave , blood pressures were averaged, and blood flows were integrated. 1,598 chest compressions were analyzed. The location of local extrema in hemodynamic parameters as a function of positive t_int values were identified recursively by dividing the range of t_int values into increasing numbers of bins and determining which bin had the highest mean value. Results: Blood flows and pressures exhibited a non-linear dependence on t_int. The maximum CPP occurred at t_int = 90 ±2.3 ms. The maximum aortic pressure occurred at t_int = 70 ±2.3 ms. The minimum right atrial pressure occurred at t_int = 280 ±2.3 ms. The maximum carotid blood flow occurred at t_int = 100 ±2.3 ms. The maximum jugular blood flow occurred at t_int = 400 ±2.3 ms. Unsynchronized chest compressions resulted in a t_int of -21 ± 170 ms. Synchronized chest compressions resulted in a t_int of 119 ± 13 ms. Conclusions: Local maxima and minima during StdCPR were identified in several hemodynamic parameters, but the extrema were not perfectly co-located. It appears that a t_int of 90-100 ms could be optimal. SyncCPR were delivered at 119 ms, which is not far from the local maxima observed for CPP and carotid blood flow.

Abstract 438: Synchronization of Chest Compressions During Pseudo Electro-Mechanical Dissociation Improves Blood Pressures Relative to Standard Chest Compressions

Introduction: The treatment of pseudo electro-mechanical dissociation (P-EMD) with standard chest compressions leads to periods where the chest compressions and heart-beat are in phase and periods where the chest compression and heart-beat are out of phase. We hypothesized that synchronized chest compressions will improve hemodynamics relative to standardized chest compressions during P-EMD. Methods: Eight animals underwent surgical preparation and were exposed to hypoxia to induce P-EMD. The treatment period was divided into eight 45 sec epochs during which the P-EMD was left untreated or was treated with standard chest compressions or chest compressions synchronized to the R-wave in the ECG. For each epoch of CPR in each animal, we measured the rate of change in the hemodynamics over the last half of the epoch using linear regression. ANOVA modeling was used to compare changes in hemodynamics as a function of treatment. Results: The rate of change in coronary perfusion pressure (CPP), minimum right atrial pressure (RAPmin), and maximum right atrial pressure (RAPmax) was larger during synchronized compressions than during standard compressions or untreated P-EMD. The rate of change in AOPmin was larger during both treatments than during untreated P-EMD. Other differences in the rate of change were not detected between standard chest compressions and untreated P-EMD. Numerical results are shown in the table below. RAPmax decreased over time, while the other measures increased. Conclusions: Synchronized chest compressions improved several blood pressure metrics over time during P-EMD, while standardized chest compressions only improved AOPmin. While some of the changes are modest, they were sustained over 20 seconds, suggesting that continued delivery of synchronized chest compressions could result in clinically meaningful improvement in blood pressures. Table 1. ANOVA comparison of blood pressures as a function of treatment

Abstract 148: The Time Interval Between the R-Wave and Maximum Aortic Pressure Affects Chest Compression Hemodynamics in a Swine Model of Pseudo Electro-Mechanical Dissociation

Introduction: The prevalence of pseudo electro-mechanical dissociation (P-EMD) as an initial cardiac arrest state is increasing. P-EMD manifests a weak ventricular contraction, which is not sufficient to sustain life. However, the presence of a weak ventricular contraction may lead to interference with or synergy with the blood flow generated by a chest compression depending on the time interval between the compression and ventricular contraction. Hypothesis: We hypothesize that the interval between a chest compression and the ventricular contraction during P-EMD will influence the hemodynamics created by the chest compression. Methods: Using our well established hypoxic P-EMD model, we measured blood pressures and ECG during mechanical chest compression (100 CPM, 2”). A nearest-neighbor analysis determined the time interval between the R-wave and the peak aortic pressure, defined as t peak AOP - t Rwave . Peak aortic pressures that had more than one R-wave nearest neighbor were excluded. 1,497 chest compressions were analyzed. Intervals were divided into quartiles, and hemodynamic parameters were compared between the quartiles using a repeated measure ANOVA with Bonferroni correction. Results: Interval (int) quartiles were defined as: Q1: int > 100 ms; Q2: 100 ms > int > 0.0 ms; Q3: 0.0ms > int > -90 ms; Q4: - 90 ms > int. Mean arterial pressures (MAP) in mmHg as a function of interval are: Q1: 33.8±0.6; Q2: 41.1±0.6; Q3: 38.3±0.6; Q4: 33.1±0.6. The MAP value for compressions with an interval in Q2 was higher than the other quartiles ( p > 0.05). Coronary perfusion pressures (CPP) in mmHg as a function of interval are: Q1: 11.7±0.5; Q2: 15.3±0.5; Q3: 15.6±0.5; Q4: 12.9±0.5. The CPP values for compressions with an interval in Q2 or Q3 was higher than the other quartiles ( p > 0.05). Conclusions: The interval between the R-wave and the peak aortic pressure generated by a chest compression has a significant effect on the resulting hemodynamics. Shorter intervals, both positive and negative are associated with improved blood pressures during resuscitation from P-EMD. These data suggest that delivery of standard CPR during P-EMD can result in a mix of effective and less effective compressions.

A physical picture for mechanical dissociation of biological complexes: from forces to free energies

Using a suitable physical model, free energy quantities for biomolecules complexes are extracted from force spectroscopy experiments.