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Background The remaining symptoms in patients with coronavirus disease 2019 (COVID‐19) treated in intensive care unit are limited described. Therefore, we assessed patient's perception of their COVID‐19 disease, stay in intensive care, and remaining symptoms three to six months after intensive care. Methods Prospective cohort study was performed in one intensive care unit of a university hospital in Sweden during the first wave. A questionnaire with open‐ended questions and closed‐ended questions was used. Data were analyzed using qualitative and quantitative content analysis and descriptive statistics. Results Out of 123 patients treated for COVID‐19, 64 answered the questionnaire 3–6 months after discharge from intensive care. Memories from illness and hospital stay revealed in three categories; awareness of the illness, losing anchor to reality and being cared for in a dynamic environment. Information was perceived as spare by 48% and they wanted the information to be more personal. The diary was perceived as personal and was received by 33% patients. The relationship with family was affected among 39% and 13% of the patients indicated that they had not resumed their daily life. A large amount, 84%, indicated that they had remaining symptoms from COVID‐19. The dominated symptoms were impaired strength and energy both physically and mentally. Conclusion Patients reported a variety of physical and mental symptoms, and revealed memories from the ICU, and specific awareness of other patients’ health. It illustrates the need for screening patients for remaining symptoms after COVID‐19 disease and ICU care and may affect resuming patients’ daily life.

Background Using cerebral oxygen venous saturation post-cardiac arrest (CA) is limited because of a small sample size and prior to establishment of target temperature management (TTM). We aimed to describe variations in jugular bulb oxygen saturation during intensive care in relation to neurological outcome at 6 months post- CA in cases where TTM 33°C was applied. Method Prospective observational study in patients over 18 years, comatose immediately after resuscitation from CA. Patients were treated with TTM 33°C M and received a jugular bulb catheter within the first 26 hours post-CA. Neurological outcome was assessed at 6 months using the Cerebral Performance Categories (CPC) and dichotomized into good (CPC 1-2) and poor outcome (CPC 3-5). Results Seventy-five patients were included and 37 (49%) patients survived with a good outcome at 6 months post-CA. No differences were found between patients with good outcome and poor outcome in jugular bulb oxygen saturation. Higher values were seen in differences in oxygen content between central venous oxygen saturation and jugular bulb oxygen saturation in patients with good outcome compared to patients with poor outcome at 6 hours (12 [8-21] vs 5 [-0.3 to 11]% P = .001) post-CA. Oxygen extraction fraction from the brain illustrated lower values in patients with poor outcome compared to patients with good outcome at 96 hours (14 [9-23] vs 31 [25-34]% P = .008). Conclusions Oxygen delivery and extraction differed in patients with a good outcome compared to those with a poor outcome at single time points. Based on the present findings, the usefulness of jugular bulb oxygen saturation for prognostic purposes is uncertain in patients treated with TTM 33°C post-CA.

Magnetic resonance imaging (MRI) of the brain including diffusion-weighted imaging (DWI) is reported to have high prognostic accuracy in unconscious post-cardiac arrest (CA) patients. We documented acute MRI findings in the brain in both conscious and unconscious post-CA patients treated with target temperature management (TTM) at 32-34°C for 24 h as well as the relation to patients' neurological outcome after 6 months.A prospective observational study with MRI was performed regardless of the level of consciousness in post-CA patients treated with TTM. Neurological outcome was assessed using the Cerebral Performance Categories scale and dichotomized into good and poor outcome.Forty-six patients underwent MRI at 3-5 days post-CA. Patients with good outcome had minor, mainly frontal and parietal, lesions. Acute hypoxic/ischemic lesions on MRI including DWI were more common in patients with poor outcome (P = 0.007). These lesions affected mostly gray matter (deep or cortical), with or without involvement of the underlying white matter. Lesions in the occipital and temporal lobes, deep gray matter and cerebellum showed strongest associations with poor outcome. Decreased apparent diffusion coefficient, was more common in patients with poor outcome.Extensive acute hypoxic/ischemic MRI lesions in the cortical regions, deep gray matter and cerebellum detected by visual analysis as well as low apparent diffusion coefficient values from quantitative measurements were associated with poor outcome. Patients with good outcome had minor hypoxic/ischemic changes, mainly in the frontal and parietal lobes.

Background Neurological outcome after cardiac arrest (CA) is difficult to predict in the acute phase. In this pilot study, we assessed blood levels of tau protein as a prognostic marker for the neurological outcome after 6 months in patients treated with hypothermia after resuscitation from CA. Methods 22 unconscious patients resuscitated after CA were treated with mild hypothermia (32–34°C) for 26 h. Blood samples were collected at 2, 6, 12, 24, 48, and 96 h after CA, and the concentration of tau protein was analyzed. Neurological outcome was assessed with the Glasgow-Pittsburgh cerebral performance category (CPC) scale at intensive care unit (ICU) discharge and after 6 months. The higher of the two CPC scores was used. Results At ICU discharge, 21/22 patients were alive, of whom 10 had a good (CPC 1–2) outcome. After 6 months, 15/22 patients were alive, of whom 14 had a good outcome. Tau protein levels were higher among those with a poor outcome at 48 h and 96 h. At 96 h sampling, tau concentration predicted a poor outcome (CPC 3–5) with a sensitivity of 71% and a specificity of 93%. Conclusions Although in a pilot study, a late increase in plasma tau protein seems to be associated with a worse outcome after hypothermia treatment after CA, although more studies are needed.

Hypothermia treatment with cooling to a body temperature of 32-34°C has been shown to be an effective way of improving neurological outcome and survival in unconscious patients successfully resuscitated after cardiac arrest (CA). The method is used clinically but there are still many questions on the biological mechanisms and on how the treatment is best performed. This thesis focuses on cerebral and haemodynamic effects of hypothermia and rewarming. A porcine model of CA was used. To shorten time to reach target temperature, induction of hypothermia, by means of infusion of 4°C cold fluid, was started already during ongoing cardiopulmonary resuscitation. The temperature was satisfactorily reduced without obvious haemodynamic disturbances. Cerebral effects of hypothermia and rewarming were studied. Microdialysis monitoring showed signs of cerebral energy failure (increased lactate/pyruvate-ratio) and excitotoxicity (increased glutamate) immediately after CA. There was a risk of secondary energy failure that was reduced by hypothermia. Intracranial pressure (ICP) increased gradually after CA irrespectively of if hypothermia was used or not. There were no indications of increasing cerebral disturbances during rewarming. Haemodynamic effects of hypothermia treatment and rewarming were examined in a study of patients successfully resuscitated after CA. Hypothermia was induced by means of cold intravenous infusion. No negative effects on the cardiovascular system were revealed. There were indications of decreased intravascular volume in spite of a positive fluid balance. Cerebral microdialysis and ICP recording were performed in four patients. All patients had signs of energy failure and excitotoxicity following CA. ICP was only exceptionally above 20 mmHg. In contrast to the experimental study indications of increasing ischemia were seen during rewarming. Glycerol had a biphasic pattern, perhaps due to an overspill of metabolites from the general circulation. As most patients become extensively anti-coagulated following CA, intracranial monitoring is not suitable to be used in routine care.

Feeding tubes are used frequently in the intensive care unit to provide enteral nutrition. For critically ill patients, enteral nutrition is preferable to parenteral in terms of cost, complication and gut mucosal maintenance. Fine bore feeding tubes are always preferred because their soft, flexible construction and narrow diameter enables these tubes to be well tolerated by patients and they rarely contribute to sinus infections or obstruction of breathing. On the other hand it is not uncommon that these tubes are misplaced in the tracheobronchial tree or the pleural cavity, especially in high-risk patients, i.e. sedated patients, patients with weak cough reflex, endotracheally intubated patients and agitated patients (1–3). Malpositioning in the peritoneal cavity or the mediastinum through gastric or esophageal perforation is also possible ( 1, 4–7); even intravascular ( 8, 9) and intracranial misplacement have been reported (10–13). The incidence of misplacement of a feeding tube is difficult to estimate because few studies have been performed. The largest study of 1100 such tubes revealed an overall malposition rate of 1.3% ( 1), but it should be mentioned that this study included only radiographically detected misplacements. Other researchers estimate the occurrence of accidental misplacement and migration out of position as high as 13% to 20% in high-risk patients ( 14, 15).

Cardiac arrest results in immediate interruption of blood flow. The primary goal of cardiopulmonary resuscitation (CPR) is to re-establish blood flow and hence oxygen delivery to the vital organs. This thesis describes different pharmacological interventions aimed at increasing cerebral blood flow during CPR and after restoration of spontaneous circulation (ROSC).In a porcine model of cardiac arrest, continuous infusion of adrenaline generated higher cortical cerebral blood flow during CPR as compared to bolus administration of adrenaline. While bolus doses resulted in temporary peaks in cerebral blood flow, continuous infusion led to a sustained increase in this flow.Administration of vasopressin resulted in higher cortical cerebral blood flow and a lower cerebral oxygen extraction ratio as compared to continuous infusion of adrenaline during CPR. In addition, vasopressin generated higher coronary perfusion pressure during CPR and increased the likelihood of achieving ROSC.Parameters of coagulation and inflammation were measured after successful resuscitation from cardiac arrest. Immediately after ROSC, thrombin-antithrombin complex, a marker of thrombin generation, was elevated and eicosanoid levels were increased, indicating activation of coagulation and inflammation after ROSC. The thrombin generation was accompanied by a reduction in antithrombin. In addition, there was substantial haemoconcentration in the initial period after ROSC.By administration of antithrombin during CPR, supraphysiological levels of antithrombin were achieved. However, antithrombin administration did not increase cerebral circulation or reduce reperfusion injury, as measured by cortical cerebral blood flow, cerebral oxygen extraction and levels of eicosanoids, after ROSC. In a clinical study, the adrenaline dose interval was found to be longer than recommended in the majority of cases of cardiac arrest. Thus, the adherence to recommended guidelines regarding the adrenaline dose interval seems to be poor.

BACKGROUND: An investigation of the free radical scavenger sodium 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) and the weak vasodilatator Tris buffer mixture (TBM) on cerebral cortical blood flow (CC ...

BACKGROUND: In experimental cardiopulmonary resuscitation (CPR) aortic balloon occlusion, vasopressin, and hypertonic saline dextran administration improve cerebral blood flow. Free radical scavenger alpha-phenyl-N-tert-butyl-nitrone (PBN) and cyclosporine-A (CsA) alleviate neuronal damage after global ischemia. Combining these treatments, we investigated neurological outcome after experimental cardiac arrest.METHODS: Thirty anesthetized piglets, randomly allocated into three groups, were subjected to 8 min of ventricular fibrillation followed by 5 min of closed-chest CPR. The combined treatment (CT) group received all the above-mentioned modalities; group B was treated with balloon occlusion and epinephrine; and group C had sham balloon occlusion with epinephrine. Indicators of oxidative stress (8-iso-PGF(2 alpha)), inflammation (15-keto-dihydro-PGF(2 alpha)), energy crisis (hypoxanthine and xanthine), and anoxia/hypoxia (lactate) were monitored in jugular bulb venous blood. Neurological outcome was evaluated 24 h after CPR.RESULTS: Restoration of spontaneous circulation (ROSC) was more rapidly achieved and neurological outcome was significantly better in the CT group, although there was no difference in coronary perfusion pressure between groups. The jugular venous PCO2 and cerebral oxygen extraction ratio were lower in the CT group at 5-15 min after ROSC. Jugular venous 8-iso-PGF(2 alpha) and hypoxanthine after ROSC were correlated to 24 h neurological outcomeCONCLUSIONS: A combination of cerebral blood flow promoting measures and administration of alpha-phenyl-N-tert-butyl-nitrone and cyclosporine-A improved 24 h neurological outcome after 8 min of experimental normothermic cardiac arrest, indicating an ongoing neuronal injury in the reperfusion phase.

Background: Intravenous administration of vasopressin during cardiopulmonary resuscitation (CPR) has been shown to improve myocardial and cerebral blood flow. Aortic balloon occlusion during CPR may also augment myocardial and cerebral blood flow and can be used as a central route for the administration of resuscitative drugs. We hypothesized that, as compared with intravenously administered vasopressin, the administration of this drug above the site of an aortic balloon occlusion would result in a greater increase in cerebral perfusion and oxygenation during CPR and after restoration of spontaneous circulation (ROSC). Methods: Twenty piglets were subjected to 5 min of ventricular fibrillation followed by 8 min of closed-chest CPR and were treated with 0.4 U kg−1 boluses of vasopressin intravenously (the IV-vasopressin group with sham aortic balloon) or above the site for an aortic balloon occlusion (the balloon-vasopressin group). The aortic balloon catheter was inflated in the latter group 1 min after commencement of CPR and was deflated within 1 min after ROSC. Systemic blood pressures, cerebral cortical blood flow, cerebral tissue pH and PCO2 were monitored continuously and the cerebral oxygen extraction ratio was calculated. Results: During CPR, arterial blood pressure and cerebral perfusion pressure were greater in the balloon-vasopressin group, as compared with the IV-vasopressin group. These pressures did not differ between the groups after ROSC. Cerebral cortical blood flow was not significantly greater in the balloon-vasopressin group during CPR, whereas significantly higher cortical blood flow levels were recorded after ROSC. Cerebral tissue pH decreased in the IV-vasopressin group during the post-resuscitation hypoperfusion period. In contrast, decreasing pressures during the hypoperfusion period did not result in increasing tissue acidosis in the balloon-vasopressin group. Conclusions: During CPR, intra-aortic vasopressin combined with aortic balloon occlusion resulted in significantly greater perfusion pressures but not in greater cerebral cortical blood flow. After ROSC, however, a greater increase in cortical blood flow was recorded in the balloon-vasopressin group, even though the aortic balloon was deflated and perfusion pressures did not differ between the groups. This suggests that vasopressin predominantly gives vasoconstrictive effects on cerebral cortical vessels during CPR, but results in cerebral cortical vasodilatation after ROSC.

Background Intranasal balloon catheters circulated with cold saline have previously been used for the induction and maintenance of selective brain cooling in pigs with normal circulation. In the present study, we investigated the feasibility of therapeutic hypothermia initiation, maintenance and rewarming using such intranasal balloon catheters with or without addition of intravenous ice-cold fluids during and after cardiac arrest treatment in pigs. Material and methods Cardiac arrest was induced in 20 anaesthetised pigs. Following 8 min of cardiac arrest and 1 min of cardiopulmonary resuscitation (CPR), cooling was initiated after randomisation with either intranasal cooling (N) or combined with intravenous ice-cold fluids (N+S). Hypothermia was maintained for 180 min, followed by 180 min of rewarming. Brain and oesophageal temperatures, haemodynamic variables and intracranial pressure (ICP) were recorded. Results Brain temperatures reductions after cooling did not differ (3.8 +/- 0.7 degrees C in the N group and 4.3 +/- 1.5 degrees C in the N+S group; P=0.47). The corresponding body temperature reductions were 3.6 +/- 1.2 degrees C and 4.6 +/- 1.5 degrees C (P=0.1). The resuscitation outcome was similar in both groups. Mixed venous oxygen saturation was lower in the N group after cooling and rewarming (P=0.024 and 0.002, respectively) as compared with the N+S group. ICP was higher after rewarming in the N group (25.2 +/- 2.9 mmHg; P=0.01) than in the N+S group (15.7 +/- 3.3 mmHg). Conclusions Intranasal balloon catheters can be used for therapeutic hypothermia initiation, maintenance and rewarming during CPR and after successful resuscitation in pigs. (Less)

Experimental studies of haemorrhagic shock have documented a superior haemodynamic response and a better outcome in female animals as compared with male controls. Such sexual dimorphism has, nevertheless, not been reported after circulatory arrest that follows exsanguination and shock. We aimed to study differences in cerebral injury markers after exsanguination cardiac arrest in pre-pubertal piglets. The hypothesis was that cerebral injury is less extensive in female animals, and that this difference is independent of sexual hormones or choice of resuscitative fluid.Thirty-two sexually immature piglets (14 males and 18 females) were subjected to 5 min of haemorrhagic shock followed by 2 min of ventricular fibrillation and 8 min of cardiopulmonary resuscitation, using three resuscitation fluid regimens (whole blood, hypertonic saline and dextran, or acetated Ringers' solution plus whole blood and methylene blue). Haemodynamic values, cellular markers of brain injury and brain histology were studied.After successful resuscitation, female piglets had significantly greater cerebral cortical blood flow, tended to have lower S-100beta values and a lower cerebral oxygen extraction ratio. Besides, in female animals, systemic and cerebral venous acidosis were mitigated. Female piglets exhibited a significantly smaller increase in neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) expression in their cerebral cortex, smaller blood-brain-barrier (BBB) disruption and significantly smaller neuronal injury.After resuscitation from haemorrhagic circulatory arrest, cerebral reperfusion is greater, and BBB permeability and neuronal injury is smaller in female piglets. An increased cerebral cortical iNOS and nNOS expression in males implies a mechanistic relationship with post-resuscitation neuronal injury and warrants further investigation.

Therapeutic hypothermia (TH) after cardiac arrest protects from neurological sequels and death and is recommended in guidelines. The Hypothermia Registry was founded to the monitor outcome, performance and complications of TH.Data on out-of-hospital cardiac arrest (OHCA) patients admitted to intensive care for TH were registered. Hospital survival and long-term outcome (6-12 months) were documented using the Cerebral Performance Category (CPC) scale, CPC 1-2 representing a good outcome and 3-5 a bad outcome.From October 2004 to October 2008, 986 TH-treated OHCA patients of all causes were included in the registry. Long-term outcome was reported in 975 patients. The median time from arrest to initiation of TH was 90 min (interquartile range, 60-165 min) and time to achieving the target temperature (or =34 degrees C) was 260 min (178-400 min). Half of the patients underwent coronary angiography and one-third underwent percutaneous coronary intervention (PCI). Higher age, longer time to return of spontaneous circulation, lower Glasgow Coma Scale at admission, unwitnessed arrest and initial rhythm asystole were all predictors of bad outcome, whereas time to initiation of TH and time to reach the goal temperature had no significant association. Bleeding requiring transfusion occurred in 4% of patients, with a significantly higher risk if angiography/PCI was performed (2.8% vs. 6.2%P=0.02).Half of the patients survived, with90% having a good neurological function at long-term follow-up. Factors related to the timing of TH had no apparent association to outcome. The incidence of adverse events was acceptable but the risk of bleeding was increased if angiography/PCI was performed.

Background and aim: Sudden cardiac arrest survivors suffer from ischaemic brain injury that may lead to poor neurological outcome and death. The reperfusion injury that occurs is associated with damaging biochemical reactions, which are suppressed by mild therapeutic hypothermia (MTH). In several studies MTH has been proven to be safe, with few complications and improved survival, and is recommended by the International Liaison of Committee on Resuscitation. The aim of this paper is to recommend clinical practice guidelines for MTH treatment after cardiac arrest from the Scandinavian Society of Anaesthesiology and Intensive Care Medicine (SSAI). Methods: Relevant studies were identified after two consensus meetings of the SSAI Task Force on Therapeutic Hypothermia (SSAITFTH) and via literature search of the Cochrane Central Register of Controlled Trials and Medline. Evidence was assessed and consensus opinion was used when high-grade evidence (Grade of Recommendation, GOR) was unavailable. A management strategy was developed as a consensus from the evidence and the protocols in the participating countries. Results and conclusion: Although proven beneficial only for patients with initial ventricular fibrillation (GOR A), the SSAITFTH also recommend MTH after restored spontaneous circulation, if active treatment is chosen, in patients with initial pulseless electrical activity and asystole (GOR D). Normal ethical considerations, premorbid status, total anoxia time and general condition should decide whether active treatment is required or not. MTH should be part of a standardized treatment protocol, and initiated as early as possible after indication and treatment have been decided (GOR E). There is insufficient evidence to make definitive recommendations among techniques to induce MTH, and we do not know the optimal target temperature, duration of cooling and rewarming time. New studies are needed to address the question as to how MTH affects, for example, prognostic factors.

Laparoscopic liver surgery is complicated due to the structure of this organ with open sinusoids. A serious disadvantage is the risk of gas embolism (GE) due to CO2 pneumoperitoneum. CO2 can enter the vascular system through a wounded vein. A common opinion is that gas fluxes along a pressure gradient, e.g. CVP-intra abdominal pressure (IAP). The occurrence of GE could also be eased by entrainment, a ‘Venturi-like’ effect, due to cyclic differences in thoracic pressure and blood flow caused by mechanical ventilation at normal frequency.The aims of these studies were to survey, in a porcine model, the influence on respiratory and haemodynamic variables by GE, to determine at what frequency, severity and duration GE occurs during laparoscopic liver resection (LLR) and whether there are methods to influence the occurrence or severity of GE.Pulmonary and circulatory variables were monitored and measured as well as continuous blood gas monitoring. Transoesophageal echocardiogram was used to identify GE and, according to the amount of bubbles in the right outflow tract of the heart, GE was graded as 0, 1 and 2. Pneumoperitoneum was created by using CO2and IAP was set to 16 mm Hg.A single bolus dose of CO2 influenced respiratory and haemodynamic variables for at least 4 h. During LLR GE occurred in 65-70% of the animals, of which the more serious caused negative influence on cardiopulmonary variables.Elevated PEEP (15 cm H2O) increased CVP but GE occurred irrespective if CVP was lower than or exceeded IAP. In two last studies, a hepatic vein was cut and left open for 3 m before it was clipped. Interestingly, no signs of GE were seen despite an open vein and IAP > CVP in 8 of 20 animals. In the last study high frequency jet ventilation was used in order to minimise the risk of entrainment. The duration of GE was shortened.The occurrence of GE seemed to be influenced by several different factors. The physiological reaction of a GE is impossible to predict for a specific patient, and depends among other factors on comorbidity, and amount, site and entrance rate of GE.

Background: Although clinical cardiopulmonary resuscitation always includes ventilation with pure oxygen, this kind of ventilation has been reported to be associated with worse neurological outcome than ventilation with air in experimental cardiopulmonary resuscitation (CPR). The aim of the present investigation was to compare the systemic oxygen uptake during experimental closed-chest CPR including ventilation with pure oxygen or ambient air and, furthermore, to elucidate possible mechanisms of action in the regulation of pulmonary gas exchange. Methods: In 24 anesthetized piglets, 2 min of induced ventricular fibrillation and no ventilation was followed by 10 min of closed-chest CPR including i.v. administration of 0.5 mg adrenaline (at 8 min), and in one of the experimental groups alkaline buffer (at 5 min). The piglets were randomly divided into 3 groups: air ventilation during the entire CPR period with saline administration (n=8), air ventilation during the entire CPR period plus tris buffer mixture (n=8), and air ventilation for 3 min followed by 100% oxygen with saline administration (n= 8). Results: In the group ventilated with air and treated with tris buffer mixture, cardiac output was significantly greater than in the group ventilated with pure oxygen. The arterial-mixed venous oxygen content difference was approximately 25% greater with pure oxygen than with air ventilation; however, there was no difference in systemic oxygen uptake. Systemic oxygen uptake increased after administration of tris buffer mixture in the group ventilated with air. Conclusions: Pulmonary hypoxic vasoconstriction appeared to be abolished during CPR including pure oxygen ventilation. Blood flow, not ventilation or pulmonary gas exchange, is the limiting factor during experimental closed-chest CPR.

Nineteen anaesthetized piglets were investigated. After catheterization and a stabilization period, ventricular fibrillation was induced with a transthoracic DC shock, after which a 10-min period of cardiopulmonary resuscitation (CPR) took place. CPR included manual chest compression and mechanical ventilation with pure oxygen. After 1 min of CPR, an infusion of alkaline buffer was begun and completed within 5 min. A total of 50 mmol of either sodium bicarbonate (n = 6) or tris buffer mixture (n = 7) were given. These two groups were compared with a third control group (n = 6) receiving the same volume of normal saline. After 8 min of CPR all animals were given 0.5 mg adrenaline i.v., and after 10 min DC shocks were used to revert the heart back to normal sinus rhythm. Our results demonstrate that blood flow and not ventilation is the limiting factor for the efficient disposal of CO2 during CPR. This also applied when the demand for CO2 transport was increased by administration of sodium bicarbonate. The respiratory exchange ratio increased 1.9-fold, indicating that the transport of carbon dioxide was less affected than that of oxygen. The estimated alveolo-arterial oxygen tension difference, shunt, and overall ventilation/perfusion ratio increased, creating an inverse hyperbolic relationship between arterial PCO2 and PO2. The difference between mixed venous and arterial PCO2 correlated well to the mixed venous PCO2, implying more efficient pulmonary elimination of PCO2 when the mixed venous PCO2 was high. Pulmonary gas exchange during CPR appears to be independent of alkaline buffer therapy in the form of sodium bicarbonate or tris buffer mixture.(ABSTRACT TRUNCATED AT 250 WORDS)