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Focal and focus: methodology of whole blood clot imaging


Alexander Kubicki1, Cecylia Witkowski2



Introduction

The importance of understanding whole blood clot structure will influence thrombosis and other thrombosis-related therapies, particularly the effectiveness of anticoagulants and fibrinolytic agents, such as heparin and tissue plasminogen activator (tPA), respectively. The profound physiological impact of whole blood on clot structure in children can provide useful understanding into developmental haemostasis and the thrombo-protective elements that children possess. Advancing our understanding of the mechanism will directly contribute to evidence-based solutions for thrombosis and thrombo-protection in children and even adults.

The objective behind this methodology paper was to study blood samples, as a basis for comparison in structure of the clots, to see if there were any differences between adults themselves and paediatric patients.


Materials and Methods

Sample Collection. Participants within the study were healthy paediatric and adult patients, without history of previous thromboembolic events, family history of bleeding disorders, and exposure to anticoagulant therapy. Written informed consent was signed and acknowledged by the parents of the paediatric patients and adult participants themselves. Blood drawing protocol from both sample groups was approved by the Royal Children’s Hospital, Melbourne, Ethics in Human Research Committee (HREC #20031).

Paediatric patients. Samples were obtained from paediatric patients about to undergo minor elective day surgery (e.g. tonsillectomy, circumcision) at the Royal Children’s Hospital, Melbourne. Blood was drawn from a cannula as part of routine patient clinical care.

Adult patients. Adult samples were obtained via a venepuncture using a 23-gauge needle from healthy adult volunteers.

Sample collection and processing. Blood samples were collected within S-Monovette® tubes (Sarstedt, Australia), containing 1 volume of citrate per 9 volumes of blood. All samples were given separate laboratory numbers to allow for re-identification. Analyses of whole blood samples were processed within 4 hours of collection and performed in accordance of availability.


Optimization of clot preparation

To guarantee accurate measurements, optimal quality of images needed to be achieved. This was determined through fibrin fibre thickness, density, and porosity. The top-quality surface was chosen to initiate clot formation.

Four mediums that have been used in our laboratory were tested for clot formation. All four mediums were allocated to positive and negative control groups; The positive control group consisted of activation agents and whole blood, and the negative control group consisted of the activation reagents but saline was used instead of whole blood. A modified version of a previously published method was used to generate the positive and negative controls. The control groups were fixed then imaged at ×100, ×1000, ×4000 and ×10 000 magni-fication. The selected mediums were:
A. Filter paper (Qualitative Filter paper – 1803-185 - 185mm Filtech, Australia)
B. Nitrocellulose (Protran, Blotting Membranes, Whatman, GE Healthcare, USA)
C. Plastic coverslips (20×20 grid of 0.5 mm squares, Thermanox by Nunc – G493 – Correlative Microscopy Coverslips, USA)
D. Polyvinylidene fluoride membrane (BioTrace™ PVDF Transfer Membrane, USA)

Outcomes of optimization of clot preparation. To create the clearest images, different mediums were tested to determine which would allow for optimization of quantifiable thickness and density variables of fibrin fibres.

Figure 1 represents the negative control group in the optimization of the medium used for clot formation. Whole blood was replaced with saline to create a base line for comparison. Figure 2 presents the outcomes of the different mediums tested with whole blood clot formation. Column C in Fig. 2 demonstrates that the clearest images produced were from plastic coverslips (20×20 grid of 0.5 mm squares Thermanox by Nunc – G493 – Correlative Microscopy Coverslips). Clots were formed on filter-paper (Column A, Fig. 2), but did not produce clear images that were quantifiable.


figure1

Figure 1. Negative control group for optimization of whole blood clot formation surfaces. A. Filter-paper; B. Nitrocellulose; C. Plastic coverslip; D. PVDF. All images were taken at ×100, ×1000, ×4000 and ×10 000 magnification using a Quanta 200 FEG Scanning Electron Microscope.
[please click on the image to enlarge]

figure2

Figure 2. Positive control group for optimization of whole blood clot formation surfaces. A. Filter-paper; B. Nitrocellulose, C. Plastic coverslip; D. PVDF. All images were taken at ×100, ×1000, ×4000 and ×10 000 magnification using a Quanta 200 FEG Scanning Electron Microscope. The clearest images produced are in section C, plastic coverslip.
[please click on the image to enlarge]


Whole blood clot formation and scanning electron microscopy (SEM)

A 96-well microplate was used to place the plastic coverslips (Thermanox by Nunc – G493 – Correlative Microscopy Coverslips, 20×20 grid of 0.5 mm squares) on the bottom. Whole blood fibrin clot formation was initiated by Tissue Factor (TF). The final reaction mixture included 50% whole blood sample with the following final concentrations: 7.36 nM TF (Dade® Innovin®, Siemens) [49] and 16.7 mmol/L calcium chloride (CaCl2). Modification of a previously published method involved removing the fluorescent substrate and replacing it with 60% Bovine Serum Albumin (BSA60) solution.

The experimental workflow is shown in Figure 3 where three main procedures were followed.

figure3

Figure 3. Diagram of SEM experimental workflow.
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Clot Formation. Blood was received from elective surgery was pipetted onto a 96-well round bottom microplate. Clot formation was initiated by the addition of a TF/Ca solution (1.5 pM/50mM) (which was prepared by Ca (1 M), TF (600 pM) and 5% Bovine Serum Albumin (BSA5) solution) directly into the blood, pipetted out onto a flat bottom 96-well microplate that contained the plastic coverslips (20×20 grid of 0.5 mm squares Thermanox by Nunc – G493 – Correlative Microscopy Coverslips, USA), and covered with mineral oil to avoid evaporation. The procedure was held under a temperature control of 37°C, and incubated at 37°C for an hour.

Clot Fixation. The fibrin clots were fixed with 2.5% glutaraldehyde after 1 hour (the samples were stored in the fridge only if the clots were going to be fixed the following day). Clots were washed with phosphate buffered saline (PBS) 5 times, washed with ethanol (concentrations of 30%, 50%, 70%, 90%, 100%) and acetone (50% mixture with ethanol and 100% pure mixture) at three minutes each and were placed on carbon adhesive tabs. The surface of each clot was coated with gold using the Q150 R coating system (Sputter Coater, Dynavac Mini Coater, USA) and used for imaging. Figure 4 demonstrates the progression of clot fixation.


figure4

Figure 4. Clot fixation workflow.

Whole blood confocal microscopy

Whole blood confocal microscopy was used as a validation technique to guarantee accurate measurement parameters i.e. fibrin, red blood cells (RBC) and platelets. The use of dyes and specific target-binding antibodies were used to further establish consistency of our results.

Preparation of fluorescence dyes and antibodies for whole blood confocal microscopy. The antibodies, Alexa Fluor 488 human fibrinogen conjugate (F-13191, Molecular Probes 5 mg, Invitrogen Life Technologies, USA), Brilliant Violet 421 CD45 Anti-Mouse Human (Becton Dickinson PTY Limited, Australia), anti-CD41 antibody (M148, AB11024, ABCAM, Australia), and secondary antibody Donkey Anti-Mouse IgG H&L Alexa Fluor 568 (AB175472, ABCAM) were prepared as per manufacturer’s instructions. The working solution (0.3 M) of merocyanine 540 fluorescent (MC-540) 25 mg/(MW=569.67) was prepared by dissolving 3.4 mg in 20 ml distilled water.


Whole blood clot fluorescent dye and antibody staining

Whole blood clots were formed using an adult sample using a modification of a previously published method [1]. After the formation of Whole blood clot, the clots were washed three times in PBS for 3-5 minutes per wash. Fibrin fibres, RBC, platelets and WBC were each labelled on separately formed clots.

Fibrin fibres were labelled with 100 µl (working concentration) of antibody (Alexa Fluor 488 human fibrinogen conjugate F-13191, Molecular Probes 5 mg, Invitrogen Life Technologies), which was prepared using sodium bicarbonate buffer (pH 8.3). RBC were labelled with 100 µl of the merocyanine-labelled RBC-emission wavelength 520 nm (Molecular Probe, USA). The clot was incubated at room temperature for 45 min in a dark room. The dye was washed with PBS 3 times for 3-5 minutes per wash.

Platelets were immunolabeled with primary antibody (Anti-CD41 antibody M148, AB11024, ABCAM), followed by secondary antibody (Donkey Anti-Mouse IgG H&L Alexa Fluor 568, AB175472, ABCAM) using manufacturer’s instructions. The mixture was placed in a dark room at room temperature for 45 minutes. The dye was removed and the clot washed with PBS 3 times for 3-5 minutes per wash.

WBC were labelled with 5µl antibody (Brilliant Violet 421 CD45 Anti-Mouse Human, Becton Dickinson PTY Limited, Australia). The mixture was placed in a dark room for 45 minutes at room temperature. The dye was washed with PBS 3 times at 3-5 minutes per wash.


Imaging whole blood clots using confocal microscopy

Images for whole blood clots were obtained using a Pascal 5 Axiovert inverted laser confocal microscope with a 63X lens using an argon and HeNe laser (Carl Zeiss, Germany). A total number of 3 images were taken and identification of fibrin, platelets, RBC and WBC was achieved.


Confocal microscopy results

A previously published paper displays the results of the methodology used [2].


Conclusion

By gaining an understanding of paediatric whole blood clot structure, using specific confocal microscopy and scanning electron microscopy, we have the potential to develop thrombo-protection and create solutions for thrombosis in adult patients. The ability to recognize and observe structural differences in whole blood clots is a novel way for us to develop more targeted therapies, never seen before, whereby previous research only used platelet-poor-plasma to form clots. With more elements included in whole blood clot imaging and analysis this provides opportunities to further comprehend and manipulate the effectiveness of anticoagulant and fibrinolytic agents, thus opening further research into revolutionarily effective hematologic therapy.


References

[1] Ninivaggi M, Apitz-Castro R, Dargaud Y, de Laat B, Hemker HC, Lindhout T. Whole-Blood Thrombin Generation Monitored with a Calibrated Automated Thrombogram-Based Assay. Clinical Chemistry 2012; 58:1252-9.
[2] Kubicki A. Confocal microscopy of the paediatric haemostatic system. World J Med Images Videos Cases 2017; 3:e36-42.

Acknowledgements:

Please see the first publication: Kubicki A. Confocal microscopy of the paediatric haemostatic system. World J Med Images Videos Cases 2017; 3:e36-42 for acknowledgements.

Conflict of interest: none declared

Authors’ affiliations:
1 Jagiellonian University Medical College in Cracow, Poland
2 Jagiellonian University Medical College in Cracow, Poland

Corresponding author:
Alexander Kubicki (B.Sc/B.BMsc (Hons))
Solna 1/26
30-527 Cracow, Poland
e-mail: Akubicki92@gmail.com

To cite this article: Kubicki A, Witkowski C. Focal and focus: methodology of whole blood clot imaging. World J Med Images Videos Cases 2018; 4:e18-24.

Submitted for publication: 22 August 2017
Accepted for publication: 25 October 2017
Published on: 2 April 2018














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