Nonconducting Tissue Research Articles

Using the impedance map to identify non-conductive structures during cavo-tricuspidal isthmus ablation

Abstract Background The ablation of the cavo-tricuspidal isthmus (CTI) is often affected by the anatomical variability of structures made of non-conductive tissue (NCS): the Eustachian valve, tricuspid valve leaflet and Tebesio's valve, the presence of which may be associated with failure of the isthmic block despite a large number of erogations and post-ablation recurrences. Since these structures have different conductive properties from atrial tissue, it is plausible that they are characterised by their own tissue impedance, and can therefore be identified through an impedance map. Objectives The objectives of the study were: 1) to characterise CTI by impedance map, in order to identify NCS; 2) to perform ablation of CTI aimed at isolating only conductive tissue by combining impedance map with the Maximum Voltage Gradient (MVG) technique, in order to evaluate the efficacy of this strategy in acute and during follow-up. Methods From 01/2021 to 09/2023 impedance map and CTI substrate map was performed, without the use of fluoroscopy, in all patients undergoing ablation of typical atrial flutter. A 15-ohm impedance reduction from the mean atrial impedance was considered the cut-off for defining the presence of NCS. Substrate maps were constructed according to the MVG technique (0.5-2.5 mV thresholds), in particular looking for high-voltage channels in which the impedance map detected the presence of NCS. Ablation was performed only in the high-voltage channels (W45, AI500). Results Our study included 48 patients with a mean age of 68±13 years, with 92% of them being men. The mean volume of the right atrium was 153 mL, and the mean number of points per map was 1205. With the use of impedence map, we were able to identify the presence of NCS in 25 patients (52%). The distribution of non conductive structures and the mean voltage by quadrants can be seen in Figures B-C. Importantly, we found that the mean number of radiofrequency applications required to achieve isthmic block was significantly lower in patients with identified NCS compared to those without (6.3 vs. 10; p=0.007). In 60% of the patients with NCS, radiofrequency was administered in high-voltage channels that were detected in the quadrant where the NCS was present (odds ratio 3.42, p=0.04). Furthermore, we successfully achieved acute isthmic blockade in all patients, and there were no instances of recurrence during a mean follow-up period of 186 days. Conclusions Our findings demonstrate that the combination of Impedence Map and the Maximum Voltage Gradient technique is an effective approach for achieving isthmic block with a reduced number of radiofrequency applications. The identification of NCS allows for a more precise search for high-voltage channels, enabling ablations to be performed in optimal contact with the tissue. This meticulous approach is crucial for the success of the procedure.

Explainable Machine Learning to Predict Anchored Reentry Substrate Created by Persistent Atrial Fibrillation Ablation in Computational Models.

Background Postablation arrhythmia recurrence occurs in ~40% of patients with persistent atrial fibrillation. Fibrotic remodeling exacerbates arrhythmic activity in persistent atrial fibrillation and can play a key role in reentrant arrhythmia, but emergent interaction between nonconductive ablation-induced scar and native fibrosis (ie, residual fibrosis) is poorly understood. Methods and Results We conducted computational simulations in pre- and postablation left atrial models reconstructed from late gadolinium enhanced magnetic resonance imaging scans to test the hypothesis that ablation in patients with persistent atrial fibrillation creates new substrate conducive to recurrent arrhythmia mediated by anchored reentry. We trained a random forest machine learning classifier to accurately pinpoint specific nonconductive tissue regions (ie, areas of ablation-delivered scar or vein/valve boundaries) with the capacity to serve as substrate for anchored reentry-driven recurrent arrhythmia (area under the curve: 0.91±0.03). Our analysis suggests there is a distinctive nonconductive tissue pattern prone to serving as arrhythmogenic substrate in postablation models, defined by a specific size and proximity to residual fibrosis. Conclusions Overall, this suggests persistent atrial fibrillation ablation transforms substrate that favors functional reentry (ie, rotors meandering in excitable tissue) into an arrhythmogenic milieu more conducive to anchored reentry. Our work also indicates that explainable machine learning and computational simulations can be combined to effectively probe mechanisms of recurrent arrhythmia.

Unique topological classification of complex reentrant atrial tachycardias enables optimal ablation strategy

Abstract Funding Acknowledgements: Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC starting grant - SMARTHEART (Nele Vandersickel) B Reentry is a common underlying mechanism of atrial tachycardia (AT). Reentry loops form around anatomical or functional 'holes' in the electrophysiological substrate and dominate the atrial cycle. Terminating a 'full' loop may cause previously 'suppressed' loops around another hole to become dominant. Interpretation of electroanatomical maps can thus be tedious: a full loop may be easily identified, but predicting suppressed loops is complex. We propose that topological classification may aid identification of suppressed loops in complex AT and improve ablation procedures. O Given a map of reentrant clinical AT, we aim to unambiguously classify the AT and suggest an operator-independent optimal ablation strategy. M The left atrium can be described topologically as a closed surface with three ‘holes’: mitral valve, left and right pulmonary veins (similar for the right atrium). Non-conductive tissue may constitute additional topological holes in the substrate. Anatomical reentry can occur around any of the holes described above. Therefore, we uniquely categorize an AT by identifying the number of holes in combination with the presence of full and suppressed loops. Fig. 1-2 shows the finite number of possible combinations for 3 and 4 holes. An optimal ablation strategy should terminate both full and suppressed loops, by connecting their corresponding holes. Directed graph mapping (DGM) is a method and software implementation to analyze arrhythmia by constructing a directed network from local activation time maps. We extended DGM to perform topological classification of AT. We hypothesize that the full reentry loops will correspond to the cycles detected by DGM in the network, while suppressed loops are characterized by activity propagating around a hole for at least 60% of its circumference. To test our algorithms, we generated > 1000 simulations corresponding to 5 different scenarios of Fig. 1-2 whereby the circumference of the suppressed loop was varied between 50% and 90% by adding slow conductive tissue. We also classified 110 clinical cases of mapped AT retrospectively and compared our suggested ablation strategy with the clinical ablation lines which ended the AT. R Terminating solely the full loops by an ablation line led to the suppressed loops creating a slower AT in 95% of simulations. Further ablation to terminate the suppressed loop ended the AT. Following our suggested ablation strategy directly terminated the AT. The ablation strategy suggested by DGM was topologically equivalent to clinically successful ablations in 90% of clinical cases. In 5 cases, DGM correctly predicted that the chosen ablation lead to a suppressed loop becoming dominant, resulting in slower AT. C We presented a unique topological classification of AT. We showed that suppressed loops play a pivotal role as not ablating them leads to slower AT. Benefits include preventing redo maps, shorter and operator independent procedures, and possibly reducing AT recurrence.

Differentiating border-zone tissue from post-infarct scar using ripple mapping during VT ablation

Abstract Funding Acknowledgements Type of funding sources: None. Background Areas of post-infarct ventricular scar and border-zone slow conduction are often highlighted on a bipolar voltage map with generalized values 0.5mV–1.5mV. The true voltage that differentiates regions of conducting from non-conducting tissue is unknown. Ripple Mapping (RM)displays allows conducting tissue to be seen as areas supporting Ripple activation, and non-conducting tissue as areas devoid of Ripple activation. Purpose We describe application of Ripple Maps to differentiate areas of scar from conducting tissue during ischemic VT ablation. Methods Dense bipolar voltage maps were created (Pentaray catheter, pacing 80-100bpm) and presented as a single value (e.g. 0.5mV-0.5mV) to binarize the color display (red and purple). RMs were superimposed on the voltage map and played above a pre-set noise threshold (>0.05mV). The voltage map mV limit was sequentially reduced ("border-zone threshold") until only those areas devoid of Ripple bars appeared red. The surrounding border-zone supporting ripple activation thus appeared purple. We performed off-line analysis of border-zone voltage thresholds from a series of RM guided VT ablations. Results 10 consecutive patients (LVEF 32.3±7.5%) with remote myocardial infarction underwent VT ablation (median 19days (IQR 8-33) since last VT). Bipolar voltage mapping (5873±2841 points, median shell area 224cm2), revealed voltages<0.5mV covered a median 11% (IQR 7-17%) of the shell. The border-zone voltage threshold was median 0.2mV (range 0.12mV - 0.3mV). Non-conducting tissue below this value covered only median 5% (IQR 3-7%) of the entire shell. VT was mappable in 4 patients, and the isthmus was bordered by tissue below the same border-zone threshold as found in normal rhythm. The border-zone was homogenized with ablation(40-50W, median 29 mins (IQR 22-33), and clinical VT was non-inducible in all, and 9 pts (91%) remain sustained VT-free at median 90-day follow-up (IQR 23-139), 2-weeks blanking period). Picture 1 presents an infero-lateral LV infarct collected in an RV paced rhythm (7340points) and displayed at conventional bipolar voltage settings 0.5-1.5mV. Tissue with voltages<0.5mV appear red and cover 30% of the total area. In this case, this border-zone voltage threshold was defined as 0.25mV. Non-conducting tissue, seen as areas devoid of ripple bars below this value, now appeared as red, and covered only 11% of the total area. Picture 2 demonstrates the morphologies of 4 poorly tolerated induced VTs during this case. Each had near perfect pacemaps to the exit sites of border-zone tissue defined using this approach, and were targets for ablation resulting in complete non-inducibility and no VT recurrence in early follow-up. Conclusion The bipolar voltage that differentiates putative scar from bordering conducting tissue is unique to each patient, and far lower than 0.5mV-1.5mV. RM presents a practical approach to visualize the border-zone activation to guide ablation.

P975Composite electroanatomical maps locate rapid activity within low voltage zones in persistent AF

Abstract Funding Acknowledgements Our research group receives an educational grant from Abbott Inc. Introduction. Outcomes from catheter ablation of persistent AF (psAF) are not favourable. The two prevailing major directions to improve success are left atrial (LA) substrate ablation, and non pulmonary vein driver ablation. In LA substrate ablation guided by intracardiac voltage, there is debate on the most fitting mapping rhythm and the appropriate cut offs for low voltage zones (LVZ). Non pulmonary vein driver ablation requires extensive experience and relies on complex pattern recognition by the operator, introducing subjectivity, that may lead to reduced reproducibility. AF drivers have been shown to localise to LVZs. We propose an objective, patient-tailored method of identifying rapid activity within LVZs to locate drivers of psAF. Methods. Eleven patients (61 ± 10.8 years of age, 9 male) undergoing first time catheter ablation for psAF were included. 3D maps were collected with a double spiral 20 pole catheter, in non-cardiac triggered mode, recording 8s segments at each bipole. Mean AF voltage (AFV) a AF cycle length (AFCL) was calculated for each 8s segment using automated algorithms. Grades of rapid activity and low voltage were defined as the 10th 20th and 30th percentile of all collected points within a patient. Percentile-matched composite LVZ-ARA maps were created on a research platform. Results. Mean LVZ percentage of the total mapped area was 4.67 ± 2.4%, 13.95 ± 3.8%, 23.81 ± 5.7% for the 10th, 20th and 30th percentiles respectively (Table 1). Mean, percentile matched LVZ-ARA overlap area percentage of the total mapped area was 0.3 ± 0.25% (10th-10th), 0.86 ± 0.58 (20th-20th), 3.1 ± 1.9% (30th-30th). ARAs represented a small proportion of all LVZs. Location of overlap areas differed significantly between patients and were marked with colours. Multi-colour areas including purple represent LVZ, multi-colour areas excluding purple, show LVZ-ARA overlap (examples in Fig 1). Conclusion. Analysis of LVZ-ARA overlap by mean AFV and AFCL provides an objective method of identifying potential drivers that localise to LVZs. The identified overlap areas constituted small, occasionally disparate areas within the LVZ of the LA. By adjusting the AFCL and AFV percentiles, the overlap areas can be tailored at the operator’s discretion, maintaining reproducible, objective decision making, without the need for complex pattern recognition. If ablation is planned, established techniques can be used to target the overlap areas, such as homogenisation or transection and connection to anatomical or ablative non-conductive tissues. AFCL 10th AFCL 20th AFCL 30th AFV 10th AFV 20th AFV 30th All patients 128 ± 13 ms 144 ± 10 ms 150 ± 9 ms 0.15 ± 0.02 mV 0.19 ± 0.03 mV 0.24 ± 0.04 mV Mean values of percentile cut offs. AFCL: AF cycle length; AFV: AF voltage Abstract Figure. Fig 1

Targeted Osmotic Lysis of Highly Invasive Breast Carcinomas Using Pulsed Magnetic Field Stimulation of Voltage-Gated Sodium Channels and Pharmacological Blockade of Sodium Pumps.

Concurrent activation of voltage-gated sodium channels (VGSCs) and blockade of Na+ pumps causes a targeted osmotic lysis (TOL) of carcinomas that over-express the VGSCs. Unfortunately, electrical current bypasses tumors or tumor sections because of the variable resistance of the extracellular microenvironment. This study assesses pulsed magnetic fields (PMFs) as a potential source for activating VGSCs to initiate TOL in vitro and in vivo as PMFs are unaffected by nonconductive tissues. In vitro, PMFs (0–80 mT, 10 msec pulses, 15 pps for 10 min) combined with digoxin-lysed (500 nM) MDA-MB-231 breast cancer cells stimulus-dependently. Untreated, stimulation-only, and digoxin-only control cells did not lyse. MCF-10a normal breast cells were also unaffected. MDA-MB-231 cells did not lyse in a Na+-free buffer. In vivo, 30 min of PMF stimulation of MDA-MB-231 xenografts in J/Nu mice or 4T1 homografts in BALB/c mice, concurrently treated with 7 mg/kg digoxin reduced tumor size by 60–100%. Kidney, spleen, skin and muscle from these animals were unaffected. Stimulation-only and digoxin-only controls were similar to untreated tumors. BALB/C mice with 4T1 homografts survived significantly longer than mice in the three control groups. The data presented is evidence that the PMFs to activate VGSCs in TOL provide sufficient energy to lyse highly malignant cells in vitro and to reduce tumor growth of highly malignant grafts and improve host survival in vivo, thus supporting targeted osmotic lysis of cancer as a possible method for treating late-stage carcinomas without compromising noncancerous tissues.

Isthmus sites identified by Ripple Mapping are usually anatomically stable: A novel method to guide atrial substrate ablation?

Postablation reentrant ATs depend upon conducting isthmuses bordered by scar. Bipolar voltage maps highlight scar as sites of low voltage, but the voltage amplitude of an electrogram depends upon the myocardial activation sequence. Furthermore, a voltage threshold that defines atrial scar is unknown. We used Ripple Mapping (RM) to test whether these isthmuses were anatomically fixed between different activation vectors and atrial rates. We studied post-AF ablation ATs where>1 rhythm was mapped. Multipolar catheters were used with CARTO Confidense for high-density mapping. RM visualized the pattern of activation, and the voltage threshold below which no activation was seen. Isthmuses were characterized at this threshold between maps for each patient. Ten patients were studied (Map 1 was AT1; Map 2: sinus 1/10, LA paced 2/10, AT2 with reverse CS activation 3/10; AT2 CL difference 50 ± 30ms). Point density was similar between maps (Map 1: 2,589 ± 1,330; Map 2: 2,214 ± 1,384; P=0.31). RM activation threshold was 0.16±0.08mV. Thirty-one isthmuses were identified in Map 1 (median 3 per map; width 27 ± 15mm; 7 anterior; 6 roof; 8 mitral; 9 septal; 1 posterior). Importantly, 7 of 31 (23%) isthmuses were unexpectedly identified within regions without prior ablation. AT1 was treated following ablation of 11/31 (35%) isthmuses. Of the remaining 20 isthmuses, 14 of 16 isthmuses (88%) were consistent between the two maps (four were inadequately mapped). Wavefront collision caused variation in low voltage distribution in 2 of 16 (12%). The distribution of isthmuses and nonconducting tissue within the ablated left atrium, as defined by RM, appear concordant between rhythms. This could guide a substrate ablative approach.

Ventricular arrhythmias and sudden cardiac death

### Key points There are 50–100 unexpected, sudden cardiac deaths (SCDs) per 100 000 population per year in Europe and USA, categorized by symptom onset to cardiac arrest time of <1 h.1 Despite the decline in coronary artery disease mortality and advancements in resuscitation services, survival from these events remains low. Unfortunately, they often occur outside the hospital environment and are associated with a survival rate of <10%. The majority occur in adults over 35 yr of age and at least half of these events can be attributed to ventricular arrhythmias, although the true incidence is unknown due to inevitable degeneration to asystole if unwitnessed. These arrhythmias may be the presenting complaint in the emergency department and may feature throughout the perioperative period, including at preoperative assessment, during surgery and in the recovery phase, for example in intensive care. They may also be the direct target of therapy in the case of electrophysiological catheter ablation and implantable cardioverter defibrillators (ICDs). They are more frequent and carry greater risks in patients with structural heart disease, but younger patients with ion-channel abnormalities can also be susceptible.2 This review classifies the causes and significance of ventricular arrhythmias based on the presence or absence of structural heart disease and provides a simple system to aid confident distinction from supraventricular arrhythmias. …

A Prospective Study of Ripple Mapping in Atrial Tachycardias: A Novel Approach to Interpreting Activation in Low-Voltage Areas.

Post ablation atrial tachycardias are characterized by low-voltage signals that challenge current mapping methods. Ripple mapping (RM) displays every electrogram deflection as a bar moving from the cardiac surface, resulting in the impression of propagating wavefronts when a series of bars move consecutively. RM displays fractionated signals in their entirety thereby helping to identify propagating activation in low-voltage areas from nonconducting tissue. We prospectively used RM to study tachycardia activation in the previously ablated left atrium. Patients referred for atrial tachycardia ablation underwent dense electroanatomic point collection using CARTO3v4. RM was played over a bipolar voltage map and used to determine the voltage "activation threshold" that differentiated functional low voltage from nonconducting areas for each map. Ablation was guided by RM, but operators could perform entrainment or review the isochronal activation map for diagnostic uncertainty. Twenty patients were studied. Median RM determined activation threshold was 0.3 mV (0.19-0.33), with nonconducting tissue covering 33±9% of the mapped surface. All tachycardias crossed an isthmus (median, 0.52 mV, 13 mm) bordered by nonconducting tissue (70%) or had a breakout source (median, 0.35 mV) moving away from nonconducting tissue (30%). In reentrant circuits (14/20) the path length was measured (87-202 mm), with 9 of 14 also supporting a bystander circuit (path lengths, 147-234 mm). In breakout tachycardias, splitting of wavefronts resulted in 2 to 4 incomplete circuits. RM-guided ablation interrupted the tachycardia in 19 of 20 cases with the first ablation set. RM helps to define activation through low-voltage regions and aids ablation of atrial tachycardias.

Mixed partial anomalous pulmonary venous drainage coexistent with an aortic valve abnormality – analysis of ultrasound diagnostics in a 10-year-old girl with Turner syndrome

The authors present a case of echocardiographic diagnosis of a rare congenital cardiovascular anomaly in the form of mixed partial anomalous pulmonary veins connection in a 10-year-old girl with Turner syndrome and congenital mild stenosis of insufficient bicuspid aortic valve, made while diagnosing the causes of intestinal tract bleeding. The article presents various diagnostic difficulties leading to the delayed determination of a correct diagnosis, resulting from the absence of symptoms of circulatory failure in the early stage of the disease and the occurrence of severe and dominant auscultatory phenomena typical for congenital aortic valve defect which effectively masked the syndromes of increased pulmonary flow. The authors discuss the role of the impact of phenotypic characteristics of the Turner syndrome, in particular a short webbed neck restricting the suprasternal echocardiographic access and the presence of psychological factors associated with a long-term illness. The importance of indirect echocardiographic symptoms suggesting partial anomalous pulmonary veins connection in the presence of bicuspid aortic valve, e.g. enlargement of the right atrium and right ventricle, and paradoxical interventricular septum motion were emphasized in patients lacking ASD, pulmonary hypertension or tricupid and pulmonary valve abnormalities. The methodology of echocardiographic examination enabling direct visualization of the abnormal vascular structures was presented. Special attention was paid to the significance of highly sensitive echocardiographic projections: high right and left parasternal views in sagittal and transverse planes with patient lying on the side, with the use of two-dimensional imaging and color Doppler. Finally, the limitations of echocardiography resulting from the visualization and tracking of abnormal vascular structures hidden behind ultrasound non-conductive tissues were indicated, as was the role of other diagnostic modalities, such as angio-CT and/or nuclear magnetic resonance.

Growth Stresses are Highly Controlled by the Amount of G-Layer in Poplar Tension Wood

To determine how gelatinous fibres and gelatinous layers contribute to the magnitude of longitudinal growth stress in tension wood, anatomical measurements of gelatinous fibres were carried out on poplar tension wood (Populus I4551). It was found that (a) no gelatinous fibres were observed under a growth strain level of 0.06 to 0.08%; (b) almost 100% of the non-conductive tissues contained gelatinous fibres above a growth strain level of 0.15 to 0.19%; and (c) the area of fibres, the area of fibres with gelatinous layers per unit of tissue area, and the thickness of the gelatinous layers predominantly influenced the magnitude of growth stress

Mechanisms, treatment, and prevention of atrial reentry tachycardia after surgery for congenital heart disease: Intracardiac mapping and ablation in Fontan patients

Mapping studies in experimental settings and in humans have demonstrated that atrial reentry tachycardias after surgery for congenital heart defects like the Fontan operation differ significantly from typical atrial flutter. These arrhythmias are based on non-conductive tissue, such as atriotomy scars, or prosthetic material, such as tubes and patches, resulting from prior cardiac surgery. Stable reentrant activation can occur around these scars or patches, as well as adjacent anatomic obstacles such as the orifices of the great veins or the atrioventricular annulus which create protected zones of atrial tissue. Full knowledge of the underlying electrophysiological and anatomical substrate is critical for delineation of the protected zones of the reentrant circuit. However, acquiring such information is often difficult, if not impossible, with traditional mapping techniques using standard multipolar electrode catheters together with pacemapping and entrainment mapping techniques, particularly in multiple and possibly intertwined circuits. To overcome these limitations novel mapping systems using electroanatomical mapping and non-contact mapping have recently been applied to these patients. The use of these novel mapping systems permits direct association of electrical activity at a particular location of myocardium with the corresponding anatomical structures, a combination which should allow for more effective targeting of RF energy applications.

SECONDARY PHLOEM ANATOMY IN CORDAITEAN AXES

Secondary phloem anatomy in cordaitean axes is described based on Carboniferous coal ball permineralizations from Kansas and Ohio. In both stems and roots, phloem consists of alternating, tangential bands of sieve cells and axial parenchyma. Sieve cells are elongate with tapered end walls and have oval‐rectangular sieve areas on their radial walls. A narrow band of tissue close to the cambium is interpreted as phloem that was probably functional at the time of deposition; the remainder of the extensive secondary phloem presumably represents nonconducting tissue and can be divided into a number of zones based on histological characters. The changes that occur from the innermost phloem out to the periphery of the stem are discussed and are compared with previous descriptions of cordaite phloem.

Application of the pipe model theory to predict canopy leaf area

The pipe model theory presents the idea that a unit weight of tree foliage is serviced by a specific cross-sectional area of conducting sapwood in the crown. Below the crown, a large fraction of the tree bole may be nonconducting tissue, so the sapwood area would have to be known to estimate foliage. We applied the pipe model theory to the analysis of several western coniferous species to learn whether the distribution of canopy leaf area could be accurately estimated from knowledge of the sapwood cross-sectional area at various heights, including breast height (1.37 m). Results are excellent, but taper in the conducting area must be considered when sapwood area is measured below the crown.

Intermediate metabolism of electric tissue in relation to function. I. Glycolytic enzymes and succinic oxidase

Studies have been initiated with the aim of obtaining more detailed information on the intermediary metabolism of electric tissue in relation to electrical activity and ion flux. The following results are described in this paper: 1. 1. Appropriate methods were worked out for preparing extracts of electric tissue and for testing them for enzyme activity. 2. 2. All the enzymes of the glycolytic scheme from phosphoglucoisomerase through lactic acid dehydrogenase have been demonstrated to be present in electric tissue extracts with the exception of triosephosphate isomerase and 1,3-phosphoglyceric kinase. 3. 3. The presence of the two latter enzymes is indicated by the experiments on glycolysis with whole homogenates. 4. 4. Some of the optimal conditions and cofactor requirements of glycolysis of electric tissue homogenates were worked out. 5. 5. Succinic oxidase activity was studied in two types of electric organ, the Bundle of Sachs and the main organ; the Q O2 average in the former is 3.5; in the latter, 2.1. 6. 6. Succinic oxidase activity of electric tissue was compared with that in other conducting tissue (striated muscle, heart) and nonconducting tissue (kidney and liver). The succinic oxidase activity in electric tissue was as high as in striated muscle, but it was still higher in heart, kidney, and liver.

The characterization of tissue cholinesterases

1. 1. Trypsin digestion does not improve the yield of cholinesterases extractable from animal tissues. 2. 2. Tissue cholinesterases can be characterized (a) by their pS-maxima and their Michaelis-Menten constants, and (b) by the ratio of the inhibitory effect of hexa- and deca-methonium at a given substrate concentration. 3. 3. It is observed that the pseudocholinesterases present in dog's serum and tissues are identical in all respects, whereas the true cholinesterases appear to differ in specificity from one organ to the other. 4. 4. It appears possible that like the brain, erythrocytes of dog and man also contain a mixture of true and pseudo cholinesterase. 5. 5. It is suggested that all cholinesterases act as enzymic buffer systems, both in conductive and non-conductive tissues.