Optimization of a widefield structured illumination microscope for non-destructive assessment and quantification of nuclear features in tumor margins of a primary mouse model of sarcoma.

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1 × 1.6 mm (3.4 mm(2)) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm(-1) used in conjunction with a 4 × 0.1 NA objective (v=0.165). This yielded an optical section thickness of 128 µm and an 8.9 × contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy

Ten‐year evolution of a massive transfusion protocol in a level 1 trauma centre: have outcomes improved?

Background: We aimed to evaluate the evolution and implementation of the massive transfusion protocol (MTP) in an urban level 1 trauma centre. Most data on this topic comes from trauma centres with high exposure to life-threatening haemorrhage. This study examines the effect of the introduction of an MTP in an Australian level 1 trauma centre. Methods: A retrospective study of prospectively collected data was performed over a 14-year period. Three groups of trauma patients, who received more than 10 units of packed red blood cells (PRBC), were compared: a pre-MTP group (2002–2006), an MTP-I group (2006–2010) and an MTP-II group (2010–2016) when the protocol was updated. Key outcomes were mortality, complications and number of blood products transfused. Results: A total of 168 patients were included: 54 pre-MTP patients were compared to 47 MTP-I and 67 MTP-II patients. In the MTP-II group, fewer units of PRBC and platelets were administered within the first 24 h: 17 versus 14 (P = 0.01) and 12 versus 8 (P < 0.001), respectively. Less infections were noted in the MTP-I group: 51.9% versus 31.9% (P = 0.04). No significant differences were found regarding mortality, ventilator days, intensive care unit and total hospital lengths of stay. Conclusion: Introduction of an MTP-II in our level 1 civilian trauma centre significantly reduced the amount of PRBC and platelets used during damage control resuscitation. Introduction of the MTP did not directly impact survival or the incidence of complications. Nevertheless, this study reflects the complexity of real-life medical care in a level 1 civilian trauma centre

Ten-year evolution of a massive transfusion protocol in a level 1 trauma centre: have outcomes improved?

Background: We aimed to evaluate the evolution and implementation of the massive transfusion protocol (MTP) in an urban level 1 trauma centre. Most data on this topic comes from trauma centres with high exposure to life-threatening haemorrhage. This study examines the effect of the introduction of an MTP in an Australian level 1 trauma centre. Methods: A retrospective study of prospectively collected data was performed over a 14-year period. Three groups of trauma patients, who received more than 10 units of packed red blood cells (PRBC), were compared: a pre-MTP group (2002–2006), an MTP-I group (2006–2010) and an MTP-II group (2010–2016) when the protocol was updated. Key outcomes were mortality, complications and number of blood products transfused. Results: A total of 168 patients were included: 54 pre-MTP patients were compared to 47 MTP-I and 67 MTP-II patients. In the MTP-II group, fewer units of PRBC and platelets were administered within the first 24 h: 17 versus 14 (P = 0.01) and 12 versus 8 (P < 0.001), respectively. Less infections were noted in the MTP-I group: 51.9% versus 31.9% (P = 0.04). No significant differences were found regarding mortality, ventilator days, intensive care unit and total hospital lengths of stay. Conclusion: Introduction of an MTP-II in our level 1 civilian trauma centre significantly reduced the amount of PRBC and platelets used during damage control resuscitation. Introduction of the MTP did not directly impact survival or the incidence of complications. Nevertheless, this study reflects the complexity of real-life medical care in a level 1 civilian trauma centre

Modulation depth as a function of grid frequency for non-scattering phantoms and scattering phantoms (two µ_s′ levels).

<p>The points labeled A and B are referenced in the Discussion section.</p

Schematic demonstrating the method used to measure the optical sectioning strength of the imaging system.

<p>Also shown is a detailed diagram of the structure of the solid phantom used for measurement. Three separate phantoms were creating with increasing levels, µ_s′ = 0, 10, 20 cm⁻¹.</p

Optical section thickness measurements from solid phantoms.

<p>(<b>A</b>) Plot of the mean image intensity as a function of distance from focal plane used to determine optical section thickness. The circles represent data that was acquired on the µ_s′ = 0 cm⁻¹ phantom using a 4×NA = 0.1 objective with an illumination frequency of 19.6 mm⁻¹. The solid line represents the Stokseth approximation (Eq. 4) calculated using the same variables. (<b>B</b>) Plot relating optical section thickness to illumination grid frequency for a range of reduced scattering coefficients. The datapoints represent the measured values on phantoms, and the solid line represent the theoretical value calculated using the Stokseth approximation. The dotted arrows show how the optical section thickness was measured on the left and how it was placed on the corresponding plot on the right.</p

Detailed schematic of the SIM imaging system.

<p>The λ_ex peak was 480 nm and the λ_em peak was 520 nm. The spatial filter diameter was adjusted to allow only the 0 and +1 diffraction orders to pass. An image of group 7 of the USAF resolution target acquired by the system is also shown.</p

Fluorescent images of mouse sarcoma tissue processed using a high-pass filter (HPF) algorithm to isolate cell nuclei.

<p>The <i>in situ</i> sectioned image (acquired using f = 31.7 mm⁻¹) closely resembles the 50 µm tissue slice image (acquired from frozen sections), while the <i>in situ</i> uniform image failed to isolate the majority of cell nuclei.</p