Counting the platelets: a robust and sensitive quantification method for thrombus formation

TL;DR: In this paper, a new method for analysis and quantification of platelet thrombus formation that can facilitate comparison of results between research groups was proposed, which was found less sensitive to microscope and image adjustments and provides more details on thrombosis development dynamics than the methods for measuring fluorescence intensity and thrombo-volume estimation.

Abstract: Flow chambers are common tools used for studying thrombus formation in vitro. However, the use of such devices is not standardised and there is a large diversity among the flow chamber systems currently used, and also in the methods used for quantifying the thrombus development. It was the study objective to evaluate a new method for analysis and quantification of platelet thrombus formation that can facilitate comparison of results between research groups. Whole blood was drawn over a collagen patch in commercial Ibid or in-house constructed PDMS flow chambers. Five percent of the platelets were fluorescently labelled and z-stack time-lapse images were captured during thrombus formation. Images were processed in a Python script in which the number of platelets and their respective x-, y- and z-positions were obtained. For comparison with existing methods the platelets were also labelled and quantified using fluorescence intensity and thrombus volume estimations by confocal microscopy. The presented method was found less sensitive to microscope and image adjustments and provides more details on thrombus development dynamics than the methods for measuring fluorescence intensity and thrombus volume estimation. The platelet count method produced comparable results with commercial and PDMS flow chambers, and could also obtain information regarding the stability of each detected platelet in the thrombus. In conclusion, quantification of thrombus formation by platelet count is a sensitive and robust method that enables measurement of platelet accumulation and platelet stability in an absolute scale that could be used for comparisons between research groups.

Summary

Introduction

• Platelets are essential to keep vascular integrity, but may at the same time cause thrombosis during pathological conditions (1) .
• This diversity is further stimulated by the use of soft lithography with polydimethylsiloxane (PDMS), which facilitates prototyping and in-house manufacturing of flow chambers (7) .
• Furthermore, thrombus volume and surface coverage measurements does not separate thrombus size increment by platelet accumulation from the parallel process of platelet-induced clot retraction, which simultaneously may reduce thrombus volume, as previously demonstrated by Ono et al. (11) .
• Apart from requiring expensive instrumentation, the image capturing process is often slower on confocal microscopes (with the exception of some spinning disc configurations), making it less suitable for fast time-lapse acquisition.
• To eliminate such user bias there are software with automated functions that makes these decisions.

Ethic Statement

• The research, blood sampling and consent procedure was approved by the local ethical review board in Linköping.
• Verbal informed consent was obtained from all blood donors, no documentation of the consent or any personal information about the blood donors was saved, thereby ensuring anonymization of the samples.

Materials

• DiOC6 were from Invitrogen Molecular Probes (Eugene, OR, USA).
• Monoclonal antibody against CD41, clone PM6/248 (preservative free) was from AbD Serotec.
• HORM Collagen was obtained from Takeda (Linz, Austria).
• Sylgard 184 base and curing agent were from Dow Corning Europe .

Blood collection and fluorescent labelling

• Whole blood was drawn from healthy volunteers into hirudin sampling tubes.
• The blood was used within 4 hours and was stored at room temperature as recommended by Roest et al. (6) .
• Directly before the flow chamber experiment, the labelled blood fraction was returned to the unlabelled fraction and gently mixed.
• 2) To determine the volume from confocal microscopy images and measure the fluorescence intensity all platelets (100%) were homogenously labelled using DiOC6 (0.25 µM).
• The PRP was reconstituted with the remaining RBC fraction before the experiments.

Flow chamber construction and experiments

• The PDMS flow chamber was moulded on a template of photo-patterned SU-8 resist on a silicon wafer.
• A collagen strip (250 µm wide) was coated on the glass slide with collagen solution (500 µg/mL), and a straight PDMS channel (height: 60 µm, width: 250 µm) was placed perpendicular over the collagen strip .
• Flow chambers were blocked with BSA (1 mg/mL) for 15 minutes prior to use.
• For comparative experiments, Ibidi Sticky-Slide I 0.1 Luer (Munich, Germany) flow chambers were mounted on collagen-coated glass slides as described above.

Image capture and analysis

• Z-stack time-lapse images were captured with a wide-field 20x objective (NA 0.8) on a Zeiss Axio Observer Z1 with a Colibri LED-module and a Neo 5.5 sCMOS camera (Andor Technology Ltd., UK) controlled by µManager software (Vale lab, UCSF).
• Confocal images were acquired with a 20x objective (NA 0.8) on a Zeiss Axio Observer LSM 700 with Zen software.
• Imaris (Bitplane AG, Switzerland) was used to render and calculate a 3D-volume from the Z-stack confocal images.
• Detailed descriptions of the image processing and analysis steps are provided in the supplemental materials and methods section, Suppl.

Using platelet count as quantification method

• Counting the number of platelets in the forming thrombus may give a more reliable measure of thrombus size and also provide additional information regarding other thrombus properties, such as platelet distribution within the thrombus and thrombus stability.
• The optimal threshold level required to distinguish the labelled platelets from the background in the image data was set using a threshold probe.
• When the optimal threshold level is found, all image data from that time series is passed through a script performing background reduction, thresholding, and finally, object counting.
• A longer distance between images in the z-axis allows for a more rapid acquisition throughout the thrombus height and therefore a shorter interval between time-points.
• The centre-of-mass calculation that is performed to return the platelet x-, y-and z-position will also be affected by an increasing distance between image planes in the z-stack.

Comparison with other techniques

• In order to test the accuracy and robustness of the presented quantification method, the authors investigated how the platelet count method compares to two other frequently used methods for quantification of thrombus formation, i.e. fluorescence intensity measurement, and thrombus volume estimation from confocal microscopy images.
• These differences become more pronounced during the later stages of thrombus build-up and the authors hypothesize that these differences may arise from an unequal contribution of fluorescence from the labelled platelets, which is dependent on the z-axis position of the accumulating platelets.
• The final microscopy image used for analysis is affected by several factors, which may or may not be within control of the user.
• The results are presented in Figure 6B and C , and it is evident that the estimated thrombus volume was considerably affected by the intensity variations for both threshold methods.
• Notably, when comparing the volume measurement with the platelet count method it becomes evident that the volume calculation is affected by the intensity variations to a much greater extent than the platelet count method, even without the use of the threshold probe.

Measuring thrombus characteristics

• The platelets and their position in the thrombus can be used for further analysis that can be valuable in determining the characteristics of the growing thrombus, in particular in terms of thrombus stability.
• To investigate these possibilities the authors performed experiments with additions of the Rho kinase inhibitor HA1077 (120 µM), which has previously been described to induce thrombus instability (11) .
• This type of measurement will of course give some information regarding the thrombus stability in terms of increasing or decreasing platelet number.
• Interestingly, from Figure 8C it is clear that an embolization event starts with platelet instability at the top of the thrombus, and within seconds this instability propagates down through the entire volume until the embolization occurs.
• Another possible extension of the method, which is not explored herein, is to acquire additional information regarding platelet activation state for each detected platelet.

Comparing different flow chambers using platelet count quantification

• To exemplify how the presented quantification method can be used for comparing results obtained in different flow chamber systems the authors preformed the same experiment in a commercial flow chamber from Ibidi and an in-house constructed PDMS flow chamber (both at shear rate 1400 s -1 ) (n=5).
• Thrombus formation was induced by a collagen patch in the flow chamber and experiments were performed with and without ADP receptor blockade by the P2Y12 inhibitor Cangrelor (5 µM).
• To enable a straightforward comparison of the two systems the thrombus development was quantified as platelet count per area (mm 2 ) of collagen coating.
• As expected the P2Y12 inhibition had a substantial impact on thrombus development , resulting in less aggregation and development of the thrombus in zaxis, which is visible in the heat maps from a single donor , presenting platelet count with time and z-axis distribution.
• These differences could be a result of dissimilar flow/shear profiles during thrombus development in the two systems since the flow chamber heights are different, and the flow rates in the chambers were set to match the shear rate at the flow chamber wall.

Conclusion

• Herein the authors introduced a method for quantifying the platelet thrombus, compatible with both wide-field and confocal microscopy.
• The authors have demonstrated that quantification performed with the platelet count method is very robust and not as easily influenced by image intensity variations as for example confocal volume measurements.
• The development of flow chamber techniques has indeed carried the research field of thrombosis and haemostasis forward.
• The great diversity in designs, quantification methods and other experimental parameters makes comparison of data between research groups problematic.
• A) The x-, y-and z-positions of all detected platelets were used to visualize the thrombus in 3D using the software Paraview™.

Figures

Figure 4. Comparison of fluorescence intensity and platelet count quantification methods. The accumulation of platelets on collagen in four individual PDMS flow chamber experiments (shear rate: 1400 s-1) was quantified using fluorescence intensity and platelet count. The blood samples were labelled both for the fluorescence intensity measurements (100% labelled platelets, DiOC6) and the platelet count method (5% labelled platelets, anti-CD41–CF555). Time-lapse z-stack images were acquired with a 20x objective (NA 0.8) fluorescence wide-field microscope. A) Total fluorescence intensity in the images was measured over time in the visual field. The baselines are affected by the initial background fluorescence (as seen in the insert figure) and normalization is therefore required. B) Platelet count of labelled platelets in the visual field performed on the same experiments as in A. C)

Figure 5. Quantitative distribution in the z-axis for different quantification methods. Platelet thrombi were formed on collagen in a PDMS flow chamber at a shear rate of 1400 s-1 with 5% labelled platelets (anti-CD41–CF555) and 100% labelled platelets (DiOC6). Z-stack images were acquired with a fluorescence wide-field microscope (20x objective, NA 0.8) and a confocal microscope (20x objective, NA 0.8). The confocal volume (DiOC6 platelets), wide-field platelet count (anti-CD41–CF555 platelets) and wide-field fluorescence intensity (DiOC6 platelets) were quantified on the same thrombus. A) The z-axis distribution for the three methods are presented as values normalized to the range 0 to 100%. Note that the captured z-stack started below the collagen coating and the thrombus starts at z-position 4 µm. B) Sample fluorescence images used for quantification by the methods in A, showing three different positions along the z-axis. The scale bar in the images represents 50 µm.

Figure 9. Platelet count used for comparing two different flow chamber systems.

Figure 8. Quantification of thrombus formation and stability. Thrombus formation on collagen was performed in a PDMS flow chamber at a shear rate of 1400 s-1, with 5% labelled platelets (anti-CD41– CF555). Time-lapse z-stack images were captured using wide-field fluorescence microscopy (20x objective, NA 0.8) and analysed with the platelet count method. Thrombus instability was induced by the addition of the Rho kinase inhibitor HA1077 (120 µM). Platelet accumulation is presented as (A) heatmap 2D histogram of platelet count in time and z-axis, and (B) plotted as total platelet count in each time-point. The number of unstable platelets was calculated and are presented as a (C) percentage of platelets in a 2D heatmap corresponding to (A), and also plotted as the overall percentage unstable platelets in each time point (D). Stable/unstable platelets were also plotted in yellow/red as time-lapse images (E) to visualize the localization of these platelets during an embolization event. The images correspond to the red markers in B and D. The arrow represents 50 µm and indicates the direction of flow.

Figure 1. Image processing steps for background reduction and determining a suitable threshold with a threshold probe. A PDMS flow chamber was used to form a thrombus on collagen with 5% labelled platelets (anti-CD41–CF555) at a shear rate of 1400 s-1. A) Original fluorescence wide-field micrographs (20x objective, NA 0.8) of the formed thrombus were processed with a difference of Gaussians algorithm for reduction of the image background. The scale bar represents 50 µm. B) The threshold level for each flow chamber experiment was determined by an automated threshold probe. The threshold probe finds the local maximum at III, which corresponds to the optimal threshold level where the number of detected platelets is maximized (insert with linear scale). C) Threshold images from different threshold levels corresponding to the numbers in the B. The scale bar represents 50 µm.

Figure 2: Visualization of thrombus formation. Platelet thrombus formation on collagen was performed in a PDMS flow chamber at a shear rate of 1400 s-1 with 5% labelled platelets (anti-CD41–CF555). Time-lapse z-stack images were captured with widefield fluorescence microscopy (20x objective, NA 0.8) and analysed with the platelet count method. A) The x-, y- and z- positions of all detected platelets were used to visualize the thrombus in 3D using the software Paraview™. The red dots represent the detected platelets and the surrounding blue spheres represent hypothetical volumes of the remaining non-labelled platelet fraction in the thrombus. B) Platelet positions were used to plot time-lapse graphs of the thrombus development viewed from

Figure 7. Direct comparison of method robustness between the confocal volume calculation (A) and the platelet count method (B). Thrombi were formed on collagen in a PDMS flow chamber (shear rate: 1400 s-1) with fluorescent labels for volume measurement (100% labelled platelets, DiOC6) and platelet count (5% labelled platelets, anti-CD41-CF555). Z-stack images were acquired with a fluorescence widefield microscope (20x objective, NA 0.8) and a confocal microscope (20x objective, NA 0.8). The z-stack images were subjected to intensity adjustments by multiplication with a factor in the range 0.92 to 1.08. Image stacks were then thresholded with a single threshold value optimized for the original image stacks (multiplier 1.00). The platelet count values are presented with and without the use of the threshold probe. All values are presented as mean ± SD of three experiments.

Figure 6. Method robustness of confocal volume estimation and platlet count quantification. Platelet thrombi were formed on collagen in a PDMS flow chamber (shear rate: 1400 s-1) with fluorescent labels for volume measurement (100% labelled platelets, DiOC6) and platelet count (5% labelled platelets, antiCD41-CF555). Z-stack images were acquired with a fluorescence wide-field microscope (20x objective, NA 0.8) and a confocal microscope (20x objective, NA 0.8). A) Impact of image processing steps on confocal volume estimation. Comparing two available threshold methods in the software Imaris; absolute intensity and background subtraction. The original confocal image, surface reconstruction and volume calculations are presented from a representative flow chamber experiment. B) Volume estimations based on confocal images were compared from images captured with different master gain setting on the microscope. The confocal image for gain 510 and surface reconstruction for gain settings 510 and 600 are presented. The grid in the imaris images represents 25x25 µm. C) Volume calculation performed in Imaris using confocal images captured with different gain settings are presented as mean ±

Figure 3. Characterizing the influence of Z-axis resolution on platelet detection

Full text

Counting the platelets: a robust and sensitive
quantification method for thrombus formation
Kjersti Claesson, Tomas Lindahl and Lars Faxälv
Linköping University Post Print
N.B.: When citing this work, cite the original article.
Original Publication:
Kjersti Claesson, Tomas Lindahl and Lars Faxälv, Counting the platelets: a robust and sensitive
quantification method for thrombus formation, 2016, Thrombosis and Haemostasis, (115), 6,
1178-1190.
http://dx.doi.org/10.1160/TH15-10-0799
Copyright: Schattauer
http://www.schattauer.de/
Postprint available at: Linköping University Electronic Press
http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-130073

1
Counting the platelets: a robust and
sensitive quantification method for
thrombus formation
Kjersti Claesson, Tomas L. Lindahl, Lars Faxälv
Department of Clinical and Experimental Medicine, Linköping University, Sweden
Corresponding author: Kjersti Claesson, Kjersti.claesson@liu.se
Linköping University
Department of Clinical and Experimental Medicine
SE-581 85 Linköping
Sweden
+46 10-103 89 37
Running title: Counting the platelets
This study was supported by a grant from the Swedish Research Council, Project No K2015-79X-22644-
01-3 and by Linköping University.

2
Summary
Flow chambers are common tools used for studying thrombus formation in-vitro. However, the use of
such devices is not standardized and there is a large diversity among the flow chamber systems
currently used, and also in the methods used for quantifying the thrombus development.
It was the study objective to evaluate a new method for analysis and quantification of platelet thrombus
formation that can facilitate comparison of results between research groups.
Whole blood was drawn over a collagen patch in commercial Ibid or in-house constructed PDMS flow
chambers. Five percent of the platelets were fluorescently labeled and z-stack time-lapse images were
captured during thrombus formation. Images were processed in a Python script in which the number of
platelets and their respective x-, y- and z-positions were obtained. For comparison with existing
methods the platelets were also labeled and quantified using fluorescence intensity and thrombus
volume estimations by confocal microscopy. The presented method was found less sensitive to
microscope and image adjustments and provides more details on thrombus development dynamics than
the methods for measuring fluorescence intensity and thrombus volume estimation. The platelet count
method produced comparable results with commercial and PDMS flow chambers, and could also obtain
information regarding the stability of each detected platelet in the thrombus. In conclusion,
quantification of thrombus formation by platelet count is a sensitive and robust method that enables
measurement of platelet accumulation and platelet stability in an absolute scale that could be used for
comparisons between research groups.

3
Introduction
Platelets are essential to keep vascular integrity, but may at the same time cause thrombosis during
pathological conditions (1). Due to this dual nature of platelets and their important function, platelets
have been thoroughly studied in different models. One approach is to utilise in-vitro flow chambers for
studies of platelet interactions, thereby attempting to replicate the process of thrombus formation
under arterial or venous shear rates in a controlled setting (2). There are both commercially available
and in-house constructed flow chambers, demonstrating a large diversity in both design and
functionality (2–6). This diversity is further stimulated by the use of soft lithography with
polydimethylsiloxane (PDMS), which facilitates prototyping and in-house manufacturing of flow
chambers (7).
Flow chambers are commonly used with image capture in both wide-field and confocal fluorescence
microscopes to detect platelet adhesion and thrombus formation. There are several methods for
quantifying the thrombus in the acquired image data, but at present with little standardization, making
comparison difficult between research groups. Commonly used quantification methods include;
measurements of fluorescence intensity (8,9), surface coverage (10) and thrombus volume estimation
based on confocal images (10,11). However, these measurements may not truly reflect the actual
accumulation of platelets. Fluorescence intensity measured in a single focal plane close to the surface
may lead to underestimation of aggregating platelets further out from the surface, since the out-of-
focus platelets contribute with less intensity compared to platelets in the focal plane. Furthermore,
thrombus volume and surface coverage measurements does not separate thrombus size increment by
platelet accumulation from the parallel process of platelet-induced clot retraction, which simultaneously
may reduce thrombus volume, as previously demonstrated by Ono et al. (11). This raises questions

4
regarding the individual contribution of these parallel processes to thrombus size when measuring
thrombus volume or surface coverage.
Thrombus volume is generally estimated from z-stack image data acquired with confocal microscopy
(5,10–12). Apart from requiring expensive instrumentation, the image capturing process is often slower
on confocal microscopes (with the exception of some spinning disc configurations), making it less
suitable for fast time-lapse acquisition. An increased temporal resolution have the benefit of capturing a
more detailed view of the of thrombus formation dynamics. Considering these limitations, non-spinning
disc confocal microscopy may be more suitable for measuring end-point results.
Another obstacle when trying to quantify thrombus volume/coverage in a standardized and
reproducible manner is that manual adjustments are often included in the end result when setting the
intensity threshold levels that separate the platelet from the background. To eliminate such user bias
there are software with automated functions that makes these decisions. However, without exact
knowledge of the underlying algorithms in such functions this will entail an uncertainty on how
variations in fluorescence intensity, from e.g. aging light sources, photo bleaching or detector gain
adjustments may translate into variations in the volume/coverage measurement.
The diversity of flow chamber design and quantification methods obstructs comparisons between
thrombus formation experiments in vitro. We propose a robust and unbiased method for quantification
of platelet thrombus formation suitable for both wide-field and confocal microscopy, based on an
algorithm for time-resolved quantification and determination of individual platelet positions within the
developing thrombus.

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Counting the platelets: a robust and sensitive quantification method for thrombus formation" ?

In this paper, the authors proposed a method for quantifying the platelet thrombus, compatible with both wide-field and confocal microscopy. 

Q2. What is the purpose of this study?

In conclusion, quantification of thrombus formation by platelet count is a sensitive and robust method that enables measurement of platelet accumulation and platelet stability in an absolute scale that could be used for comparisons between research groups. 

Q3. What was the purpose of the study?

It was the study objective to evaluate a new method for analysis and quantification of platelet thrombus formation that can facilitate comparison of results between research groups.