Introduction
Studying the architecture of platelets and fibrin fibres is done using scanning electron microscopy. Fibrin fibre structural arrangement in thrombus was first demonstrated in 1981 using electron microscopy (Weisel et al., 1981). Thrombus structure was later described using iridium labelled platelets in a small cohort of patients (Badimon et al., 1987). Microscopic mechanical properties of thrombus were first described by Collet et al using electron microscopy (Collet et al., 2005). This was found to be time consuming and error prone, hence the current focus is mainly on fibrin structure derived from platelet poor plasma. However, the quantitative assessment of thrombus using SEM is still in early stages of development and is not yet formally standardised. From our laboratory, we have recently published ultrastructural differences in fibrin fibres of formed thrombus using scanning electron microscopy comparing T2DM and non-DM patients following NSTE-ACS (Viswanathan et al., 2014).
Principles and technique of scanning electron microscopy
Light microscopes cannot generate images of magnification higher than 1000x. Hence scanning electron microscopy (SEM) was used. SEM is a type of electron microscope that produces images of a sample by scanning it with a focused bean of electrons. SEM uses the principles of signal generation when high voltage electron beams are focused on a smaller area on the surface of the solid biological sample. The electrons interact with atoms in the sample, producing various signals that can be detected and that contain information about the sample's surface topography / external morphology, composition, crystalline structure and orientation of individual subunits of the sample. The electron beam is generally scanned in a raster scan pattern, and the beam's position is combined with the detected signal to produce an image. SEM can achieve resolution better than 1 nanometer. Specimens can be observed in high vacuum, in low vacuum, in wet conditions (in environmental SEM), and at a wide range of cryogenic or elevated temperatures. The electron beam receiver collects data over a selected area of the surface of the sample, and a 2-dimensional image is generated that displays spatial variations in these properties. Areas ranging from 1 cm to 5 microns in width can be imaged using conventional SEM techniques (magnification ranging from 20x to approximately 30,000x, spatial resolution of 50 to 100 nm).
112 Components of SEM include:
i) electron optical column - source to produce electrons ii) electromagnetic coils to control and modify the beam iii) vacuum systems - “holds” vacuum to minimise artefacts
iv) Signal detection & display unit - consists of detectors that collect the signal and electronics to produce an image from the signal.
Detailed principles and capacities involved in the image acquisition and analysis are beyond the scope of this thesis. Basically, in SEM accelerated electrons carry significant amounts of kinetic energy, and when these focused electrons hit and decelerate from the surface of the sample, the energy is dissipated as various signals. The types of signals include secondary electrons (SE), back-scattered electrons (BSE), characteristic X-rays, cathodoluminescence (CL), specimen current and transmitted electrons. Back-scattered electrons (BSE) are beam electrons that are reflected from the sample by elastic scattering. A backscatter electron detector covers the trajectory of the backscattered electrons. Secondary electrons and backscattered electrons can be efficiently detected separately at a low accelerating voltage and the detector is placed in such a way to avoid the pathway of the primary electron beam. Images from backscattered and secondary electrons can be of high resolution can be obtained.
Transmission EM (TEM) is another high resolution EM and can achieve magnification of up to 450,000x. I decided to use SEM rather than TEM for the following reasons:
i. SEM is better suited to study the thrombus formed in Badimon chamber as TEM may focus only on a very small area of the thrombus ii. thrombus preparation on a very thin slice of aorta will be technically
challenging for TEM
iii. fibrin to fibre interactions are better studied using SEM
iv. cross sectional imaging of thrombus by TEM will be very time consuming and less likely to yield more information
SEM study protocol
With the help of the senior operators at Electron Microscopy services, Newcastle University, I performed a series of pilot image acquisition under supervision as a part of my training. For better reproducibility and to improve processing speed, the pilot
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image was standardised at approximately 60x magnification using an 8 kilo Voltage electron beam and the sample kept at 6mm distance from the electron gun. For high power 3,200x magnification and for ultrahigh power 15,000x magnification were chosen as standard for this study.
I decided to analyse 60 random patient samples (20 in each group) on both visits. The samples were identified by the study identification numbers, chosen randomly by an independent member of our team. This kept me blinded to the group the particular study identification number belonged to during the analysis. In order to detect a 10% change in fibrin diameter from baseline 1 week after clopidogrel therapy with 80%
power, we estimated that a sample size of 17 patients per group was required. Hence,
I chose my sample size as 20 per group. Each patient had two high shear aorta specimens.
Thrombus laden aorta samples from the Badimon chambers were fixed in 2% glutaraldehyde. A sample of 1mm width was taken from each chamber (1x low shear and 2x high shear) at the median point of the aorta. Only high shear thrombus specimens were used for analysis. After 72 hours of fixing, the thrombus specimens were serially treated with varied concentration of ethyl alcohol. After critical point drying, the samples were mounted using silver adhesive. They were then gold coated in a specialised chamber and the samples were loaded to SEM. Standard pilot images were taken at 60x magnification. Six images were taken at high power 3.2x103
magnification and further six images were taken at 15x103 magnification. The areas of
interest were identified using a validated random grid model using 2x3 squares (Silvain
et al., 2011). Total numbers of fibres were counted in a square grid model with area of
36sq.microns. All the images were analysed by me and I was blinded from which group the study identification number belonged to.
The images were analysed using Image ProPlus® software 4.0.1 (Media Cybernetics Inc, MD, USA). I first focused on the standard pilot image (60x magnification) and identified platelet rich areas and fibrin areas using a simple grid analysis model and calculated proportional content of each of them. This did not provide the absolute value of platelet or fibrin content of thrombus but percentage area occupied by each of the components. Internal validation was performed and intra observer CV (KB) was 12.4% and inter-observer CV (KB vs KW) was 16.5% for platelet-fibrin content of thrombus.
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I then proceeded with high power images of 3.2x103 magnification and focused on two
distinct structures, platelets (disc shaped) and fibrin (filament shaped)
Platelets in the sample were identified by the following validated structural features, Figure 3.6 (Williams text book of Haematology, 8th edition, McGraw Hill, New York,
USA):
i. disc shaped structure ii. presence of pseudopodia iii. Presence of filaments
iv. Presence of exosomal globular structures (exocytosis of golgi apparatus and granules)
v. Presence of clumping
Fibrin fibres were identified by the following validated features, Figure 3.7: i. Long filamentous architecture
ii. Presence of interlinkage between filaments iii. Twisting of filaments
iv. Attachment to platelets v. Entrapment of platelets
The “hub and spokes” model of thrombus assessment was created following the discussion with the senior laboratory technicians. It was hypothesised in our previous work that the presence of higher number of “hub and spokes” would limit the energy to be used in individual fibrin fibre polymerisation and thus make them more susceptible for autolysis.
Dendrite identification algorithm of Image Proplus® version 4.0 was used to analyse the fibrin to fibrin interlinkage. This software identified fibrin filaments in the thrombus and was used to count the numbers and measure the size of individual fibres. The dendrite function algorithm specifically identifies fibrin-fibrin interlinkage in a predefined area of thrombus. The “hub and spokes model” was applied on the image to study individual fibrin to fibrin interaction.
“Hub” was defined as presence of three or more individual fibrin fibres intersecting each other at three different angles with at least 20 degrees in between. Those with angles less than 20 were defined as parallel fibrin fibres. This model helped us to study
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the compactness of the thrombus. Individual fibrin fibres were known as “spokes”. As the thrombus was heterogeneous in any given field in SEM, I also measured clusters of hub and spokes per square micron of the field. This provided a quantitative assessment of compactness of the thrombus.
For quality control, every fourth sample was re-analysed and if the differences in fibrin diameter or platelet diameter were more than 10%, analysis was performed for the third time and the results of the third analysis was included in the analysis.
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Figure 3.6 Electron microscopy appearance of platelets
Electron microscopy appearance of platelets in various stages of activation was seen at high power magnification (x3600) and at 8kV energy. Activated platelets were characterised by the presence of pseudopodia seen as an “end on” appearance. Granules were seen as small projections on the surface of the platelets. The platelets were seen here at various stages of changes in shape. The fibrin filaments are seen bridging the adjacent platelets, thereby resulting in platelet clumps.
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Figure 3.7 Electron microscopic appearance of fibrin fibres
Fibrin fibres viewed at ultra-high power magnification by scanning electron microscopy. Fibrin fibres were arranged in longitudinal pleated structure with trapped RBC’s and platelets. The presence of hub and spoke appearance is shown in this section with lateral fibrin fibre interlinkages.
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Step by step SEM analysis protocol
SEM analysis of the thrombus consisted of three distinct stages (sample preparation, image acquisition and image analysis), as describe below:
Sample preparation
1. After completion of Badimon chamber study, the aorta substrate was carefully removed from the chamber
2. The middle 1mm width of aorta laden with thrombus was dissected using a precision blade/scalpel. Care was taken not to disturb freshly formed thrombus. The same was repeated for the two other chambers, so that three specimens were obtained (1x low shear, 2x high shear). The samples were immediately fixed in 2% glutaraldehyde in Sorensons phosphate buffer
3. The specimens were then stored at 5°C for at least 72 hours
4. The fixed thrombus samples were then rinsed twice for 15 minutes using 0.01M phosphate buffer solution
5. This was followed by step by step dehydration using various strengths of ethanol as follows: (gradual increase in concentration to minimise artefacts):
a) 25% ethanol for 30 minutes b) 50% ethanol for 30 minutes c) 75% ethanol for 30 minutes d) 100% ethanol one hour e) 100% ethanol for one hour
6. This was followed by critical point drying using Baltec critical point dryer. 7. Samples were then mounted on an aluminium stub with sample number written
on the bottom surface and Acheson silver Electrodag was used as an adhesive on the adventitial side of the aorta
8. Using Poloron SEM coating unit, with argon vapour, colloid gold was spray coated (up to 15nm thickness) on the surface of the thrombus
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Image acquisition
1. Maximum of six gold coated samples in the aluminium stubs were loaded in the SEM tray
2. One tray was then loaded in to the holder and the door is closed 3. Electron detector was then adjusted
4. SEM machine was then switched on to generate vacuum
5. The green flicker from tungsten filament was adjusted to sharpen the image 6. Image detector was switched on and the sample image appeared on the screen 7. Recommended settings used to obtain a magnification of 60-72x times as a basal
image
8. Image was acquired to the folder in the PC using Olympus software 9. This image was the pilot image and used to check patients ID number
10. Major grid was used and the image was divided to 6 squares. One image was obtained from each major grid
11. Image was refocused to the square of interest starting with left top square
12. This focused square was then divided to 4 squares using a high power grid. One square was again chosen at random and focused at 3,200 to 3,600x magnification. Further fine tuning of the image was performed to obtain better contrast. Image was then acquired and stored in the PC
13. The high power image was further divided into 4 squares using ultra power grid. One square was chosen at random and focused at approximately 15,000x magnification. The image was fine-tuned, acquired and stored in PC as in step 8 14. Then returned back to low power at 60-70x magnification and moved to the next
square in pilot grid, bottom left corner. Steps repeated as above
15. Image acquisition was repeated from middle top, middle bottom, right top and right bottom squares. One high power and one ultra-high power image for each major square in the pilot grid was available
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16. Returned back again to low power at 60-70x magnification and moved to the next square in pilot grid, bottom left corner. Steps repeated as above
17. Image acquisition was repeated from middle top, middle bottom, right top and right bottom squares. One high power and one ultra-high power image for each major square in the pilot grid was available
18. In total each sample had six high power (x3200 magnification) and six ultra-high power (x15000 magnification). There were four samples for each patients (2 from visit 1 and 2 from visit 2) generating 24 samples for each patient
19. Once completed, images were backed up in the portable hard drive 20. Filament was turned down, and SEM left under vacuum
Image analysis
The images were analysed using Image ProPlus® software 4.0.1 (Media Cybernetics Inc, MD, USA) as explained in the previous section.
121 Figure 3.8 Image acquisition in SEM – Major Grid
The sample was divided into 6 squares using major grid. One square was chosen at random and further divided into four high power grid (squares). One square was chosen again at random from the high power grid, for high power image (3200x - 3600x magnification). One high power image per one major grid of the pilot image. Similar steps were repeated for acquiring ultra-power images (15000x magnification) per one major grid of pilot image. Solid arrows indicate the area of interest in the pilot image of high power grid and ultra-high power grid.
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3.4.3 Thromboelastography® and Platelet Mapping™ - assessment of