Noninferior Red Cell Concentrate Quality after Repeated Air Rescue Mission Transport for Prehospital Transfusion.

Background: Transfusion of red cell concentrates (RCCs) is an integral therapy after severe hemorrhage or trauma. Prehospital transfusion offers an immediate intervention in emergency cases. Air ambulance-based prehospital transfusion, already used in different countries, is currently established in Germany. Limited information is available for regulatory-compliant transport logistics of RCCs and their quality after repeated air rescue missions. Thus, the aim of this study was (i) to validate regulatory-compliant logistics and (ii) to assess product quality, analyzing biochemical parameters and RBC morphology. Study Design and Methods: Due to regulatory requirements, we adapted a rotation system of 1 day transport, 1 day quarantine storage and 1 day storage over the entire RCC shelf life. RCCs transported on air rescue missions (flight group) were compared against a control group, treated identically except for helicopter transport. RCCs were visually inspected, and their temperature was documented throughout the entire rotation cycles. RCCs at the end of shelf life (end point samples) were assessed for levels of hemoglobin, hematocrit, free hemoglobin, hemolysis, mean corpuscular volume, potassium and pH. In addition, morphological changes were assessed using flow morphometry. Results: In total 81 RCCs were assessed in the flight group and 50 in the control group. Within the flight group, 30 RCCs were transfused. RCCs were dispatched on average 11 times (7-13 times). The average flight time was 18.3 h (6.6-28.8 h). The rotation system ensured adherence to regulatory guidelines, especially compliance to storage conditions of +2 to +6°C of intermediate storage. Biochemical and morphological quality parameters did not exhibit any changes upon repeated air rescue missions. A correlation with respect to the flight time was not observed either. Discussion: The quality of RCCs after repeated air rescue missions is noninferior to control samples regarding biochemical and morphological parameters. The product quality is within German regulations for up to 42 days of storage. The logistics and maintenance of the thermal conditions are safe and feasible. Thus, a rotation system of RCCs offers a regulatory-compliant option to supply air rescue missions with RCCs to allow life-saving prehospital transfusions at the incident scene.

Flow morphometry to assess the red blood cell storage lesion.

We present a novel automated system for morphology analysis of red blood cells (RBC) under flow. RBC concentrates collected by blood banks for transfusions are stored for periods of up to several weeks, during which time a number of changes occur, collectively termed the storage lesion. Typically the extent of hemolysis is the defining criterion to determine the acceptability of the RBCs for transfusions. Morphological changes are related with biochemical alteration during the storage of RBCs. The typical blood smear procedure for determining such changes is a labor-intensive and potentially biased manual process. The advantage of the flow morphometry system presented here is that it provides fully automated morphological classification of RBCs with large sample numbers in a short time. Our system uses a commercially available flow cell and flow conditions that prevent adhesion of RBCs, thus eliminating the need for blocking agents such as albumin that affect the distribution of cell shapes. Our morphometry results are validated by comparison with standard biochemical assays (hemolysis, ATP) for blood from 17 donors stored under blood bank conditions for 13 weeks. We show that the percentage of spherocytes present can be used to estimate the status of RBC concentrates. © 2017 International Society for Advancement of Cytometry.

Image processing for identification and quantification of filamentous bacteria in in situ acquired images.

BACKGROUND: In the activated sludge process, problems of filamentous bulking and foaming can occur due to overgrowth of certain filamentous bacteria. Nowadays, these microorganisms are typically monitored by means of light microscopy, commonly combined with staining techniques. As drawbacks, these methods are susceptible to human errors, subjectivity and limited by the use of discontinuous microscopy. The in situ microscope appears as a suitable tool for continuous monitoring of filamentous bacteria, providing real-time examination, automated analysis and eliminating sampling, preparation and transport of samples. In this context, a proper image processing algorithm is proposed for automated recognition and measurement of filamentous objects. METHODS: This work introduces a method for real-time evaluation of images without any staining, phase-contrast or dilution techniques, differently from studies present in the literature. Moreover, we introduce an algorithm which estimates the total extended filament length based on geodesic distance calculation. For a period of twelve months, samples from an industrial activated sludge plant were weekly collected and imaged without any prior conditioning, replicating real environment conditions. RESULTS: Trends of filament growth rate-the most important parameter for decision making-are correctly identified. For reference images whose filaments were marked by specialists, the algorithm correctly recognized 72 % of the filaments pixels, with a false positive rate of at most 14 %. An average execution time of 0.7 s per image was achieved. CONCLUSIONS: Experiments have shown that the designed algorithm provided a suitable quantification of filaments when compared with human perception and standard methods. The algorithm's average execution time proved its suitability for being optimally mapped into a computational architecture to provide real-time monitoring.

In situ microscopy as a tool for the monitoring of filamentous bacteria: a case study in an industrial activated sludge system dominated by M. parvicella.

The present study demonstrates the application of in situ microscopy for monitoring the growth of filamentous bacteria which can induce disturbances in an industrial activated sludge process. An in situ microscope (ISM) is immersed directly into samples of activated sludge with Microthrix parvicella as dominating species. Without needing further preparatory steps, the automatic evaluation of the ISM-images generates two signals: the number of individual filaments per image (ISM-filament counting) and the total extended filament length (TEFL) per image (ISM-online TEFL). In this first version of the image-processing algorithm, closely spaced crossing filament-segments or filaments within bulk material are not detected. The signals show highly linear correlation both with the standard filament index and the TEFL. Correlations were further substantiated by comparison with real-time polymerase chain reaction (real-time PCR) measurements of M. parvicella and of the diluted sludge volume index. In this case study, in situ microscopy proved to be a suitable tool for straightforward online-monitoring of filamentous bacteria in activated sludge systems. With future adaptation of the system to different filament morphologies, including cross-linking filaments, bundles, and attached growth, the system will be applicable to other wastewater treatment plants.

In situ microscopic cytometry enables noninvasive viability assessment of animal cells by measuring entropy states.

Current state of the art to determine the viability of animal cell suspension cultures is based on sampling and subsequent counting using specific staining assays. We demonstrate for the first time a noninvasive in situ imaging cytometry capable of determining the statistics of a morphologic transition during cell death in suspension cultures. To this end, we measure morphometric inhomogeneity--defined as information entropy--in cell in situ micrographs. We found that the cells are partitioned into two discrete entropy states broadened by phenotypical variability. During the normal course of a culture or by inducing cell death, we observe the transition of cells between these states. As shown by comparison with ex situ diagnostics, the entropy transition happens before or while the cytoplasmatic membrane is loosing its ability to exclude charged dyes. Therefore, measurement of morphometric inhomogeneity constitutes a noninvasive assessment of viability in real time.

Morphometric quantification of a pseudohyphae forming Saccharomyces cerevisiae strain using in situ microscopy and image analysis.

Yeast morphology and counting are highly important in fermentation as they are often associated with productivity and can be influenced by process conditions. At present, time-consuming and offline methods are utilized for routine analysis of yeast morphology and cell counting using a haemocytometer. In this study, we demonstrate the application of an in situ microscope to obtain a fast stream of pseudohyphae images from agitated sample suspensions of a Saccharomyces cerevisiae strain, whose morphology in cell clusters is frequently found in the bioethanol fermentation industry. The large statistics of microscopic images allow for online determination of the principal morphological characteristics of the pseudohyphae, including the number of constituent cells, cell-size, number of branches, and length of branches. The distributions of these feature values are calculated online, constituting morphometric monitoring of the pseudohyphae population. By providing representative data, the proposed system can improve the effectiveness of morphological characterization, which in turn can help to improve the understanding and control of bioprocesses in which pseudohyphal-like morphologies are found.

Online monitoring of the morphology of an industrial sugarcane biofuel yeast strain via in situ microscopy.

In industrial yeast fermentation processes, single-cell yeast suspensions are usually preferable to cells in aggregates, as single cells exhibit a larger contact area with the nutrient medium, which in many cases helps optimize the process. In addition to affecting fermentation time and efficiency, cell aggregates (e.g., pseudohyphal yeast morphology) may also impair centrifugation systems, one of the most expensive and complex steps of the production process that involves the recovery of yeast cells for subsequent fermentation cycles. To date, no standard technique allows for a systematic diagnosis of yeast morphology in real time during sugarcane biofuel fermentation. Accordingly, we investigate an in situ microscope (ISM) for online monitoring of the density and morphology of an industrial Saccharomyces cerevisiae strain widely used in Brazilian distilleries (PE-2). During batch and repeated batch sugarcane molasses fermentation, the instrument revealed single cells, budding yeast cells, and pseudohyphae, all in a variety of sizes and shapes. The ISM image analysis indicated that the volume of single yeast cells increased by roughly 40% over the lag phase before budding and remained approximately constant thereafter. Pseudohyphae with three and more cells appeared mostly during the stationary phase. Cooling problems were simulated by raising the temperature from 33 to 45 °C. During this thermal stress, single cells as well as budding cells and pseudohyphae forming cells became smaller and exhibited intracellular inhomogeneities. From these results, we conclude that an ISM is a useful tool for monitoring yeast morphology during sugarcane fermentation. Atypical morphologies can be detected early and be used as an automatic warning system.

In situ microscopy using adjustment-free optics.

In the past years, in situ microscopy has been demonstrated as a technique for monitoring the concentration and morphology of moving microparticles in agitated suspensions. However, up until now, this technique can only achieve a high resolution if a certain manual or automated effort is established for continuous precise focusing. Therefore, the application of in situ microscopes (ISMs) as sensors is inhibited in the cases where unattended operation is required. Here, we demonstrate a high-resolution ISM which, unlike others, is built as an entirely rigid construction, requiring no adjustments at all. This ISM is based on a specially designed water immersion objective with numerical aperture = 0.75 and a working distance of 15 µm. The objective can be built exclusively from off-the-shelf parts and the front surface directly interfaces with the moving suspension. We show various applications of the system and demonstrate the imaging performance with submicron resolution within moving suspensions of microorganisms.

In situ microscopy: a perspective for industrial bioethanol production monitoring.

This work reviews the state-of-the-art in image-based in situ methods with regard to their potential use for fermentation of Saccharomyces cerevisiae in sugarcane wine. The integration of real time information from fermentation tanks in the control strategies has high potential to promote better fermentative performance. While several image-based techniques for the measurement of cell concentration have been established, a reliable and consistent viability measurement still remains a challenging task. Reagent-free methods that estimate viability from information contained in micrograph images are reviewed. Nevertheless, the inherent complexity of the sugarcane syrup medium imposes extra challenges regarding its representation in microscopic images and their evaluation by real time image analysis.