High-Throughput Griess Assay of Nitrite and Nitrate in Plasma and Red Blood Cells for Human Physiology Studies under Extreme Conditions.

The metabolism of nitric oxide plays an increasingly interesting role in the physiological response of the human body to extreme environmental conditions, such as underwater, in an extremely cold climate, and at low oxygen concentrations. Field studies need the development of analytical methods to measure nitrite and nitrate in plasma and red blood cells with high requirements of accuracy, precision, and sensitivity. An optimized spectrophotometric Griess method for nitrite-nitrate affords sensitivity in the low millimolar range and precision within ±2 µM for both nitrite and nitrate, requiring 100 µL of scarcely available plasma sample or less than 50 µL of red blood cells. A scheduled time-efficient procedure affords measurement of as many as 80 blood samples, with combined nitrite and nitrate measurement in plasma and red blood cells. Performance and usefulness were tested in pilot studies that use blood fractions deriving from subjects who dwelt in an Antarctica scientific station and on breath-holding and scuba divers who performed training at sea and in a land-based deep pool facility. The method demonstrated adequate to measure low basal concentrations of nitrite and high production of nitrate as a consequence of water column pressure-triggered vasodilatation in deep-water divers.

Correction to Mathieu D, Marroni A, Kot J: Tenth European Consensus Conference on Hyperbaric Medicine: recommendations for accepted and non-accepted clinical indications and practice of hyperbaric oxygen treatment. Diving Hyperb Med. 2017 Mar;47(1):24-32.

Consistent with the Committee on Publication Ethics guidelines, we the above authors are initiating a partial retraction and correction of our paper: Mathieu D, Marroni A, Kot J: Tenth European Consensus Conference on Hyperbaric Medicine: recommendations for accepted and non-accepted clinical indications and practice of hyperbaric oxygen treatment. Diving Hyperb Med. 2017 Mar;47(1):24-32. We wish to make the following statement: "Regardless of the strict process of editing and proof-reading of tables included in the above-mentioned publication, we received some comments from readers which showed us that imperfect layout of Table 1 and incorrect layout of Table 2 changed significantly the conclusions which could be drawn from them. Table 1 described the relation between strength of recommendations given by the Jury of the Consensus Conference and the level of evidence based on the GRADE system. There should be a clear and straight relation showing that Level 1 "strong recommendation" should be based on GRADE A "high level of evidence (LOE)", Level 2 "weak recommendation" should be based on GRADE B "moderate LOE", Level 3 "neutral recommendation" should be based on GRADE C "low LOE" and finally no recommendation should be given when only GRADE D "very low LOE" are present. Note that there is no change to the content of the table, but only visual representation of this relationship. Table 2 has been incorrectly printed. In fact, there is no GRADE A LOE. All X marks placed in the column A should be moved to the right, to GRADE B LOE. In the same way, all X marks placed in the column B should be moved to the right, to GRADE C LOE. We voluntarily retract these tables from the above-mentioned publication, expressing our regret for the situation."

ECHM Consensus Conference and levels of evidence - Reply.

Dr Sherlock asks for clarification on the approach adopted by the European Committee on Hyperbaric Medicine (ECHM) to assessing evidence for establishing indications for hyperbaric oxygen treatment (HBOT). Firstly, regardless of the strict process of editing and proof-reading of tables included in the above-mentioned publication, we received comments from some readers that identified imperfect layout of Table 1 and incorrect layout of Table 2 which significantly changed the conclusions to be drawn from them. This concerned both the details of the methodology used and description of the ECHM recommendations and associated levels of evidence. Therefore, those tables are republished in their correct forms in this issue, hoping that this will explain at least some of the doubts and misunderstandings. Both the Editor and ourselves apologise for these errors of publication. Secondly, in the ECHM Consensus Conference methodology, we scored the evidence for clinical studies requiring double-blind randomised controlled trials (RCT) as Level A and B when, at the same time, some scoring scales require simply 'RCT', as correctly pointed out by Dr Sherlock. Long experience in organising evidence based medicine (EBM) meetings and discussions has taught us that RCTs that are not double blinded are often criticised as having serious potential bias and so are denied as level A evidence. Although we acknowledge that double blinding a clinical study in HBOT is a source of difficulty, we chose a priori to consider only double-blinded RCTs in our grading scale to avoid endless discussions about this potential bias. We are well aware that doing so means that Level A evidence is a difficult target for the hyperbaric community. We agree that many evidence scoring systems have a low level of inter-observer agreement. This is why we treat the Consensus Conference as a valuable tool that provides a better opportunity for discussing the evidence than analysis by a small group of 'experts'. This is because the whole process is transparent and available to all participants' comments and input. The final process of voting by the audience after the general discussion thus truly reflects the position of the professional hyperbaric community in Europe on the issued recommendations. By these two mechanisms, the blind application of disputable evidence scoring systems may be avoided or, at least, decreased. Thirdly, the problem of 'sham' treatments in hyperbaric research has been raised. While this has been discussed many times in the past, hyperbaric research is not the sole field where such sham treatment raises some difficulty. Surgery is probably the best example where RCTs with control arms utilising sham surgical procedures (possibly including the administration of anaesthesia) are rare and can raise major ethical problems. Nevertheless, from an EBM viewpoint, the difficulty of designing a double-blind study is never taken into account during evaluation of clinical studies. Finally, Dr Sherlock pointed out her doubts on the recommendations issued by the ECHM on idiopathic sudden sensorineural hearing loss (ISSHL). While there is no possibility to cite here the full experts' report on that issue presented during the conference, we understand that a detailed report from the Conference is being prepared for publication. In brief, the strength of evidence has been scored as Level B, in general agreement with the last Cochrane review and the UHMS Committee report. Based on this level of evidence, the Type 1 recommendation was issued with the agreement of the large majority of the Consensus Conference participants.

Tenth European Consensus Conference on Hyperbaric Medicine: recommendations for accepted and non-accepted clinical indications and practice of hyperbaric oxygen treatment.

The tenth European Consensus Conference on Hyperbaric Medicine took place in April 2016, attended by a large delegation of experts from Europe and elsewhere. The focus of the meeting was the revision of the European Committee on Hyperbaric Medicine (ECHM) list of accepted indications for hyperbaric oxygen treatment (HBOT), based on a thorough review of the best available research and evidence-based medicine (EBM). For this scope, the modified GRADE system for evidence analysis, together with the DELPHI system for consensus evaluation, were adopted. The indications for HBOT, including those promulgated by the ECHM previously, were analysed by selected experts, based on an extensive review of the literature and of the available EBM studies. The indications were divided as follows: Type 1, where HBOT is strongly indicated as a primary treatment method, as it is supported by sufficiently strong evidence; Type 2, where HBOT is suggested as it is supported by acceptable levels of evidence; Type 3, where HBOT can be considered as a possible/optional measure, but it is not yet supported by sufficiently strong evidence. For each type, three levels of evidence were considered: A, when the number of randomised controlled trials (RCTs) is considered sufficient; B, when there are some RCTs in favour of the indication and there is ample expert consensus; C, when the conditions do not allow for proper RCTs but there is ample and international expert consensus. For the first time, the conference also issued 'negative' recommendations for those conditions where there is Type 1 evidence that HBOT is not indicated. The conference also gave consensus-agreed recommendations for the standard of practice of HBOT.

Continuous real-time monitoring and recording of glycemia during scuba diving: pilot study.

INTRODUCTION: Insulin-dependent diabetes has been considered a scuba diving contraindication. This is currently being reconsidered for well-controlled diabetes. We developed a real-time continuous glucose monitor (CGM) to check glycemia, or blood glucose (BG), during diving, both for prospective studies and to increase diabetic diver safety, allowing for real-time control of glycemia and hypoglycemia prevention. To ensure CGM measurement accuracy we tested the method under hyperbaric conditions. MATERIALS AND METHODS: Two experienced diabetic divers were studied during a one-week diving cruise. BG was monitored every five minutes on every dive, by a dedicated CGM, and values were visible to the divers throughout their dives. The mean of relative difference (MRD) between CGM and capillary blood glucose was calculated. Measurement accuracy was assessed according to ISO guideline 15197 and by Clarke Error Grid (CEG) analysis. RESULTS: Both divers showed gradual BG decrease during diving. Hyperbaric chamber accuracy tests showed two of 26 MRD values (7.7%) slightly exceeding the ISO-15197 allowed difference (5%). However, our data suggest that this discrepancy may have been an artefact. DISCUSSION: Our data (even limited to two subjects only) agree with the current literature showing that also in our investigated subjects diving does not imply significant risks of hypoglycemia. The use of a real-time CGM by diabetic divers during their dives can provide immediate information on BG values and trends, thus significantly improving diving safety. The accuracy tests comparing continuous glucose monitoring (CGM) and capillary blood glucose measurement (CBM) data recorded under hyperbaric conditions showed that data recorded under pressure are very close to the ISO-15197 and CEG acceptable limits.

Decompression illness medically reported by hyperbaric treatment facilities: cluster analysis of 1929 cases.

INTRODUCTION: The term decompression illness (DCI) describes maladies resulting from inadequate decompression, but there is little consensus concerning clinically useful DCI subclasses. Our aim was to explore an objective DCI classification using multivariate statistics to assess naturally associated clusters of DCI manifestations. We also evaluated their mapping onto other DCI classifications and investigated the association with therapeutic outcome. METHODS: We defined the optimal number of clusters using "two-step" cluster analysis and Bayesian information criterion with confirmation by hierarchical clustering with squared Euclidian distances and Ward's method. The data were 1929 DCI cases reported by hyperbaric chambers to the Divers Alert Network (DAN America) from 1999-2003. RESULTS: Four robust and highly significant clusters of DCI manifestations were demonstrated containing 300, 741, 333, and 555 patients. Each cluster had characteristic manifestations. Cluster 1 was effectively pain only. For Cluster 2, characteristic manifestations included numbness, paresthesia, and decreased skin sensitivity; for Cluster 3, malaise, paralysis, muscular weakness, and bladder-bowel dysfunction; and for Cluster 4, hearing loss, localized skin swelling, tinnitus, skin rash and mottling, confusion, dyspnea/chokes, muscular problems, vision problems, altered consciousness, headache, vertigo, nausea, fatigue, dizziness, and abnormal sensations. DISCUSSION: Internal reliability was confirmed by arbitrarily dividing the dataset into two parts and repeating the analysis. The clusters mapped poorly onto traditional DCI categories (AGE, Type I DCS, Type II DCS), but more specifically onto the Perceived Severity Index (PSI). All three classification methods (DCI, Cluster, PSI) predicted complete relief of manifestations equally well. We conclude that cluster analysis is an objective method for classifying DCI manifestations independent of clinical judgment.

Serum erythropoietin levels in healthy humans after a short period of normobaric and hyperbaric oxygen breathing: the "normobaric oxygen paradox".

Renal (peritubular) tissue hypoxia is a well-known physiological trigger for erythropoietin (EPO) production. We investigated the effect of rebound relative hypoxia after hyperoxia obtained under normo- and hyperbaric oxygen breathing conditions. A group of 16 healthy volunteers were investigated before and after a period of breathing 100% normobaric oxygen for 2 h and a period of breathing 100% oxygen at 2.5 ATA for 90 min (hyperbaric oxygen). Serum EPO concentration was measured using a radioimmunoassay at various time points during 24-36 h. A 60% increase (P < 0.001) in serum EPO was observed 36 h after normobaric oxygen. In contrast, a 53% decrease in serum EPO was observed at 24 h after hyperbaric oxygen. Those changes were not related to the circadian rhythm of serum EPO of the subjects. These results indicate that a sudden and sustained decrease in tissue oxygen tension, even above hypoxia thresholds (e.g., after a period of normobaric oxygen breathing), may act as a trigger for EPO serum level. This EPO trigger, the "normobaric oxygen paradox," does not appear to be present after hyperbaric oxygen breathing.