In vivo quantification of rolling and adhered leukocytes in human sepsis.

BACKGROUND: The use of in vivo videomicroscopy at the bedside has demonstrated microcirculatory flow disturbances in sepsis. The ability of in vivo videomicroscopy to detect changes in the prevalence of rolling and adhered leukocytes that occur in sepsis is not well-described in humans. We sought to (1) develop methodology for accessing and quantifying sublingual leukocyte rolling and adherence with sidestream dark field (SDF) imaging; (2) compare the number of rolling and adhered leukocytes between patients with septic shock and non-infected controls; and (3) compare the number of rolling and adhered leukocytes between survivors and non-survivors of septic shock. METHODS: We included adult (age > 18 years) patients in the emergency department presenting with septic shock prospectively enrolled in the ProCESS trial. We recruited comparison non-infected patients as emergency department controls. Using a SDF videomicroscope, we obtained image sequences from the sublingual mucosa, quantifying rolling and adhered leukocytes per 1 mm × 1 mm visual field in a standardized 3-s clip. We report data as median and interquartile range and depicted as box plots. We compared groups using the Mann-Whitney U test, considering a p value < 0.05 significant. RESULTS: We included a total of 64 patients with septic shock and 32 non-infected controls. The median number of adhered leukocytes per field in the sepsis group was 1.0 (IQR 0-3.5) compared to 0 (0-0) in the non-infected group (p < 0.001). The median number of rolling leukocytes was 26 (10.3-42) in the sepsis group and 9.8 (4.8-17.3) in the non-infected group (p < 0.001) per field. Among the patients with sepsis (n = 64), there was an increased number of adhered leukocytes in non-survivors compared to survivors (3.0 (1-5.5) vs. 1.0 (0-3.0)) (p < 0.05); however, there was no difference in rolling leukocytes (35 (20-48) vs. 26 (10-41)) (p = 0.31). CONCLUSIONS: Our results demonstrated a higher number of rolling and adhered leukocytes in patients with septic shock when compared to non-infected controls, and an increased number of adhered leukocytes in non-survivors. TRIAL REGISTRATION: ClinicalTrials.gov , NCT00793442 ; Registered on 19 November 2008 PG0GM076659 (US NIH Grant/Contract). First submitted 18 July 2007. First posted 2 August 2007.

Microcirculatory perfusion disturbances in septic shock: results from the ProCESS trial.

BACKGROUND: We sought to determine the effects of alternative resuscitation strategies on microcirculatory perfusion and examine any association between microcirculatory perfusion and mortality in sepsis. METHODS: This was a prospective, formally designed substudy of participants in the Protocolized Care in Early Septic Shock (ProCESS) trial. We recruited from six sites with the equipment and training to perform these study procedures. All subjects were adults with septic shock, and each was assigned to alternative resuscitation strategies. The two main analyses assessed (1) the impact of resuscitation strategies on microcirculatory perfusion parameters and (2) the association of microcirculatory perfusion with 60-day in-hospital mortality. We measured sublingual microcirculatory perfusion using sidestream dark field in vivo video microscopy at the completion of the 6-h ProCESS resuscitation protocol and then again at 24 and 72 h. RESULTS: We enrolled 207 subjects (demographics were similar to the overall ProCESS cohort) and observed 40 (19.3%) deaths. There were no differences in average perfusion characteristics between treatment arms. Analyzing the relationship between microcirculatory perfusion and mortality, we found an association between vascular density parameters and mortality. Total vascular density (beta = 0.006, p < 0.003), perfused vascular density (beta = 0.005, p < 0.04), and De Backer score (beta = 0.009, p < 0.01) were higher overall in survivors in a generalized estimating equation model, and this association was significant at the 72-h time point (p < 0.05 for each parameter). CONCLUSIONS: Microcirculatory perfusion did not differ between three early septic shock treatment arms. We found an association between microcirculatory perfusion parameters of vascular density at 72 h and mortality. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00510835 . Registered on August 2, 2007.

A guide to human in vivo microcirculatory flow image analysis.

Various noninvasive microscopic camera technologies have been used to visualize the sublingual microcirculation in patients. We describe a comprehensive approach to bedside in vivo sublingual microcirculation video image capture and analysis techniques in the human clinical setting. We present a user perspective and guide suitable for clinical researchers and developers interested in the capture and analysis of sublingual microcirculatory flow videos. We review basic differences in the cameras, optics, light sources, operation, and digital image capture. We describe common techniques for image acquisition and discuss aspects of video data management, including data transfer, metadata, and database design and utilization to facilitate the image analysis pipeline. We outline image analysis techniques and reporting including video preprocessing and image quality evaluation. Finally, we propose a framework for future directions in the field of microcirculatory flow videomicroscopy acquisition and analysis. Although automated scoring systems have not been sufficiently robust for widespread clinical or research use to date, we discuss promising innovations that are driving new development.

Impact of transfusion of autologous 7- versus 42-day-old AS-3 red blood cells on tissue oxygenation and the microcirculation in healthy volunteers.

BACKGROUND: Stored red blood cells (RBCs) accumulate biochemical and biophysical changes. Maximum storage duration is based on acceptable in vitro characteristics and 24-hour survival, but not RBC function. Relatively little is known about the impact of RBC storage duration on oxygenation and the microcirculation. STUDY DESIGN AND METHODS: Eight healthy subjects donated a double RBC apheresis, which were prestorage leukoreduced and processed in AS-3. Subjects were transfused 1 unit of RBCs at 7 and 42 days after blood collection. Measurements of percentage of tissue oxygenation in the thenar eminence muscle (StO2) and brain (SctO2) were recorded with Food and Drug Administration-cleared noninvasive devices. Sublingual microvascular blood flow (microcirculatory flow index [MFI]) was quantified before and after RBC transfusion using a video microscope. Raw electronic data for all measurements were analyzed by a blinded observer at a core laboratory. RESULTS: The only pre- versus posttransfusion change observed in measurements of SctO2, StO2, or MFI was a very small increase in SctO2, from 70.4 to 71.8 (means, p=0.032) at 7 days. There was no significant difference in the amount of pre-post change at 7 days versus 42 days for any of the measures. CONCLUSION: Transfusion of 1 unit of 42-day-stored RBCs to healthy subjects has no overt detrimental effect on tissue oxygenation or the microcirculation assessed by clinically available monitors.

The impact of red blood cell storage duration on tissue oxygenation in cardiac surgery.

OBJECTIVE: Although storage alters red blood cells, several recent, randomized trials found no differences in clinical outcomes between patients transfused with red blood cells stored for shorter versus longer periods of time. The objective of this study was to see whether storage impairs the in vivo ability of erythrocytes to traverse the microcirculation and deliver oxygen at the tissue level. METHODS: A subset of subjects from a clinical trial of cardiac surgery patients randomized to receive transfusions of red blood cells stored ≤10 days or ≥21 days were assessed for thenar eminence and cerebral tissue hemoglobin oxygen saturation (StO2) via the use of near-infrared spectroscopy and sublingual microvascular blood flow via side-stream darkfield videomicroscopy. RESULTS: Among 55 subjects, there was little change in the primary endpoint (thenar eminence StO2 from before to after transfusion of one unit) and the change was similar in the 2 groups: +1.7% (95% confidence interval, -0.3, 3.8) for shorter-storage and +0.8% (95% confidence interval, -1.1, 2.9) for longer-storage; P = .61). Similarly, no significant differences were observed for cerebral StO2 or sublingual microvascular blood flow. These parameters also were not different from preoperatively to 1 day postoperatively, reflecting the absence of a cumulative effect of all red blood cell units transfused during this period. CONCLUSIONS: There were no differences in thenar eminence or cerebral StO2, or sublingual microcirculatory blood flow, in cardiac surgery patients transfused with red blood cells stored ≤10 days or ≥21 days. These results are consistent with the clinical outcomes in the parent study, which also did not differ, indicating that storage may not impair oxygen delivery by red blood cells in this setting.

Lactate Clearance in Septic Shock Is Not a Surrogate for Improved Microcirculatory Flow.

BACKGROUND: Failure to normalize lactate is associated with poor outcomes in septic shock. It has been suggested that persistently elevated lactate may result from regional ischemia due to disturbed and/or heterogenous microcirculatory blood flow. OBJECTIVES: The goal of this study was to determine if lactate clearance (LC) may serve as a surrogate marker for changes in microcirculatory blood flow in patients with septic shock. METHODS: This was a prospective observational study performed within a previously published clinical trial of l-carnitine for the treatment of vasopressor-dependent septic shock. Intravital video microscopy was performed at enrollment and 12 hours later, and microcirculatory flow index (MFI) was assessed. Associations between enrollment MFI, lactate, and Sequential Organ Failure Assessment (SOFA) score were determined, in addition to associations between ∆MFI, LC, and ∆SOFA. A preplanned subgroup analysis of only patients with an elevated initial lactate was performed. RESULTS: We enrolled a total of 31 patients, 23 with survival and sufficient quality videos both at enrollment and at 12 hours. ∆MFI, LC, and ∆SOFA were 0.1 (interquartile range [IQR] = 0 to 0.3), 18% (IQR = -10% to 46%), and -2 (IQR = -4 to 0). Both ∆MFI and LC were associated with ∆SOFA (ß = -5.3, p = 0.01; and ß = -3.5, 0.047), but not with each other, even in the subgroup of patients with an initially elevated lactate. CONCLUSIONS: We observed no association between degree of LC and change in microcirculatory blood flow in patients with septic shock. These data suggest against the hypothesis that LC may be used as a surrogate marker of microcirculatory blood flow.

The microcirculation image quality score: development and preliminary evaluation of a proposed approach to grading quality of image acquisition for bedside videomicroscopy.

PURPOSE: Side-stream dark-field microscopy is currently used to directly visualize sublingual microcirculation at the bedside. Our experience has found inherent technical challenges in the image acquisition process. This article presents and assesses a quality assurance method to rate image acquisition quality before analysis. MATERIALS AND METHODS: We identified 6 common image capture and analysis problem areas in sublingual side-stream dark-field videos: illumination, duration, focus, content, stability, and pressure. We created the "Microcirculation Image Quality Score" by assigning a score of optimal (0 points), suboptimal but acceptable (1 point), or unacceptable (10 points) to each category (for further details, go to http://www.MicroscanAnalysis.blogspot.com). We evaluated 59 videos from a convenience sample of 34 unselected, noncritically ill emergency department patients to create a test set. Two raters, blinded to each other, implemented the score. Any video with a cumulative score of 10 or higher (range, 0-60) was considered unacceptable for further analysis. RESULTS: We created the Microcirculation Image Quality Score and applied it to 59 videos. For this particular set of 59 videos, the mean (SD) passing quality score was 1.68 (0.90), and the mean (SD) failing quality score was 15.74 (6.19), with 27 of 59 passing the quality score less than 10. Highest failure occurred from pressure artifact. The interrater agreement for acceptability was assessed using Cohen κ for each category: illumination (κ = 1.0), duration (κ = 1.0), focus (κ = 0.91), content (κ = 0.76), stability (κ = 0.71), and pressure (κ = 0.82) and overall pass-fail rates (score >10) (κ = 0.66). CONCLUSION: Our Microcirculation Image Quality Score addresses many of the common areas where video quality can degrade. The criteria introduced are an objective way to assess the quality of image acquisition, with the goal of selecting videos of adequate quality for analysis. The interrater reliability results in our preliminary study suggest that the Microcirculation Image Quality Score is reasonably repeatable between reviewers. Further assessment is warranted.