This research was funded by the Science and Technology Bureau of Xiamen City (3502Z20227197), and the Science and Technology Bureau of Fujian Province (No. 2019J01070, No.2021Y0027).

The research was performed in compliance with the institutional guidelines of the Medical Ethics Committee of Huaqiao University. The cerebral blood clots were surgically removed and collected at Quanzhou First Hospital, affiliated to Fujian Medical University, with informed consent from the patients.
1. Blood clot pretreatment
<ol>
	<li>Place the clots on a clean dish, add 5 mL of physiological saline with a tweezer, shake the dish gently, and remove the saline with a pipette. 
	NOTE: Repeat the rinse three times.</li>
	<li>Cut the clots into smaller pieces (smaller than 5 mm x 5 mm x 5 mm) with a scissor. Add 5 mL of physiological saline, shake the dish gently, and remove the saline with a pipette. 
	NOTE: Repeat the rinse three times. Ensure no clot pieces are aspirated during the rinse.</li>
	<li>Transfer the clots to another clean U-plate. 
	&#8203;NOTE: The protocol can be paused here (store the plate at 2-8 &#176;C) and restarted later.</li>
</ol>
2. Thrombolysis
<ol>
	<li>Prepare the sFE working solution by adjusting the concentration of sFE to 2000 U/mL with physiological saline. 
	NOTE: The sFE was prepared according to the well-established protocols of Tang Lab15.</li>
	<li>Add 300 &#181;L of sFE working solution into the pretreated clots.</li>
	<li>Perform the first round of degradation.
	<ol>
		<li>Incubate the mixture of sFE and clots at 37 &#176;C for 0.5 h. 
		NOTE: The blot treated with physiological saline was set as negative control. Do not rotate the sample on the shaker.</li>
		<li>Mix the sample gently. Transfer the liquid part to another clean tube with a clean pipette. 
		NOTE: The remaining clots were used for another round of degradation.</li>
		<li>Centrifuge the liquid part at 200 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Transfer the supernatant to another clean tube with a pipette to obtain the recovered sFE solution.</li>
		<li>Mix the cell precipitation with 50 &#181;L of physiological saline gently. 
		NOTE: The cell mixture was stored 2-8 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Perform subsequent degradation.
	<ol>
		<li>Degrade the remaining clots (step 2.3.2) with the recovered sFE solution (step 2.3.4) at 37 &#176;C for 0.5 h. 
		NOTE: The rest steps were the same as the first degradation round. The degradation process was finished until the whole clots were degraded.</li>
	</ol>
	</li>
	<li>Perform cell collection.
	<ol>
		<li>Prepare the blood cell sample by collecting the cell mixture of each round of degradation.</li>
	</ol>
	</li>
</ol>
3. Wright&#39;s staining
<ol>
	<li>Perform cell smear.
	<ol>
		<li>Adjust cells to a density of 1 x 106 cells/mL.</li>
		<li>Add 5 &#181;L of the cells to the poly-L lysine-coated glass slide, then smear them using the cover slip.</li>
		<li>Evaporate the liquid at room temperature.</li>
	</ol>
	</li>
	<li>Perform the staining.
	<ol>
		<li>Add 200 &#181;L of Wright&#39;s dye (see Table of Materials) to the cell smear gently, and add 600 &#181;L of Wright&#39;s dye B to mix evenly.&#160;&#160;Stain for 10 min at room temperature.</li>
		<li>Rinse the dye gently with clean water. 
		NOTE: The stained cell smear can be stored at room temperature for later use.</li>
	</ol>
	</li>
	<li>Perform microscopic imaging.
	<ol>
		<li>Examine the stained cells using a light microscope (see Table of Materials).</li>
	</ol>
	</li>
</ol>
4. Routine blood examination
<ol>
	<li>Adjust cells to a density of 1 x 106 cells/mL.</li>
	<li>Analyze the cell components such as platelets, erythrocytes, white blood cells, lymphocytes, neutrophils, monocytes, eosinophils, basophils, and immature granulocytes using an autohematology analyzer according to the manufacturer&#39;s instructions (see Table of Materials).</li>
</ol>

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

<ol>
	<li>Park, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Edoxaban+versus+dual+antiplatelet+therapy+for+leaflet+thrombosis+and+cerebral+thromboembolism+after+TAVR:+The+ADAPT-TAVR+Randomized+clinical+trial.">Edoxaban versus dual antiplatelet therapy for leaflet thrombosis and cerebral thromboembolism after TAVR: The ADAPT-TAVR Randomized clinical trial.</a> Circulation. 146 (6), 466-479 (2022).</li><li>Devasagayam, S., Wyatt, B., Leyden, J., Kleinig, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+venous+sinus+thrombosis+incidence+is+higher+than+previously+thought:+a+retrospective+population-based+study.">Cerebral venous sinus thrombosis incidence is higher than previously thought: a retrospective population-based study.</a> Stroke. 47 (9), 2180-2182 (2016).</li><li>Sacco, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+updated+definition+of+stroke+for+the+21st+century:+a+statement+for+healthcare+professionals+from+the+American+Heart+Association/American+Stroke+Association.">An updated definition of stroke for the 21st century: a statement for healthcare professionals from the American Heart Association/American Stroke Association.</a> Stroke. 44 (7), 2064-2089 (2013).</li><li>Thalin, C., Hisada, Y., Lundstrom, S., Mackman, N., Wallen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+villains+and+targets+in+arterial,+venous,+and+cancer-associated+thrombosis.">Neutrophil extracellular traps: villains and targets in arterial, venous, and cancer-associated thrombosis.</a> Arteriosclerosis Thrombosis and Vascular Biology. 39 (9), 1724-1738 (2019).</li><li>Mocsai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+novel+functions+of+neutrophils+in+immunity,+inflammation,+and+beyond.">Diverse novel functions of neutrophils in immunity, inflammation, and beyond.</a> Journal of Experimental Medicine. 210 (7), 1283-1299 (2013).</li><li>Dhanesha, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKM2+promotes+neutrophil+activation+and+cerebral+thromboinflammation:+therapeutic+implications+for+ischemic+stroke.">PKM2 promotes neutrophil activation and cerebral thromboinflammation: therapeutic implications for ischemic stroke.</a> Blood. 139 (8), 1234-1245 (2022).</li><li>Ducroux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombus+neutrophil+extracellular+traps+content+impair+tpa-induced+thrombolysis+in+acute+ischemic+stroke.">Thrombus neutrophil extracellular traps content impair tpa-induced thrombolysis in acute ischemic stroke.</a> Stroke. 49 (3), 754-757 (2018).</li><li>Solomon, C., Ranucci, M., Hochleitner, G., Schochl, H., Schlimp, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+methodology+for+calculating+platelet+contribution+to+clot+strength+(platelet+component)+in+thromboelastometry+and+thrombelastography.">Assessing the methodology for calculating platelet contribution to clot strength (platelet component) in thromboelastometry and thrombelastography.</a> Anesthesia and Analgesia. 121 (4), 868-878 (2015).</li><li>Daraei, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+fiber+diameter+and+porosity+measurements+of+plasma+clots+in+scanning+electron+microscopy+images.">Automated fiber diameter and porosity measurements of plasma clots in scanning electron microscopy images.</a> Biomolecules. 11 (10), 1536 (2021).</li><li>Abbasi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+thrombus+composition+in+thrombectomy+stroke+patients+with+longer+time+to+recanalization.">Diverse thrombus composition in thrombectomy stroke patients with longer time to recanalization.</a> Thrombosis Research. 209, 99-104 (2022).</li><li>C W Francis, a., Marder, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concepts+of+clot+lysis.">Concepts of clot lysis.</a> Annual Review of Medicine. 37 (1), 187-204 (1986).</li><li>Xu, R., Ma, G., Chen, L., Cui, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Preparation and application of natural fibrinolytic enzyme from peanut worm. , (2019).</li><li>Fotso Fotso, A., Drancourt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Diagnosis+of+tick-borne+african+relapsing+fevers:+latest+developments.">Laboratory Diagnosis of tick-borne african relapsing fevers: latest developments.</a> Front Public Health. 3, 254 (2015).</li><li>Liou, G. Y., Byrd, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+bioliquid+markers+for+pancreatic+cancer:+What+we+have+vs.+what+we+need.">Diagnostic bioliquid markers for pancreatic cancer: What we have vs. what we need.</a> Cancers (Basel). 15 (9), 2446 (2023).</li><li>Tang, M., Lin, H., Hu, C., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+a+fibrinolytic+enzyme+from+Sipunculus+nudus.">Affinity purification of a fibrinolytic enzyme from Sipunculus nudus.</a> Journal of Visualized Experiments. 196, e65631 (2023).</li><li>Ge, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+antithrombotic+protease+from+marine+worm+Sipunculus+Nudus.">A Novel antithrombotic protease from marine worm Sipunculus Nudus.</a> International Journal Of Molecular Sciences. 19 (10), 3023 (2018).</li><li>Talukder, M. A., Menyuk, C. R., Kostov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinguishing+between+whole+cells+and+cell+debris+using+surface+plasmon+coupled+emission.">Distinguishing between whole cells and cell debris using surface plasmon coupled emission.</a> Biomedical Optics Express. 9 (4), 1977-1991 (2018).</li><li>Shapiro, D. J., Hicks, L. A., Pavia, A. T., Hersh, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+prescribing+for+adults+in+ambulatory++care+in+the+USA,+2007-09.">Antibiotic prescribing for adults in ambulatory care in the USA, 2007-09.</a> Journal of Antimicrobial Chemotherapy. 69 (1), 234-240 (2014).</li></ol>

The authors have no conflicts of interest to disclose.

sFE is a fibrinolytic agent that can degrade the fibrin directly and effectively12,16. Here, sFE was employed to degrade the cross-linked fibrin of the cerebral blood clots, release the enclosed cells within the clots, and analyze the cell components of the clots qualitatively and quantitatively. The microscopy data and routine blood examination indicated that the enclosed cells were released from the blood clots. Furthermore, the cell types and structures of the...

Cerebrovascular disease is one of three major diseases that can threaten human health, among which ischemic cerebrovascular disease accounts for more than 80%. Cerebral thrombosis and cerebral vein thrombosis are the most concerned ischemic cerebrovascular diseases today, mainly caused by cerebral blood clots1,2. If the treatment is not done properly, it will have high disability and mortality rates and a high recurrence rate after discharge3.
Recently, a growing number of studies have shown that the cell components of cerebral blood clots are tightly correlated with the diagnosis, treatment, and prognosis of cerebral thrombosis4,5,6. Therefore, the availability of data on thrombus composition, especially the cell components, is important for clinical diagnosis and treatment. Unfortunately, the currently available methods cannot comprehensively analyze the blood clot component quantitatively and qualitatively. For example, Martius Scarlett Blue based in-situ staining can only study the red/white blood cells of certain slices of the clot7. Immunohistochemistry (IHC) based in-situ staining can only study limited blood components of certain slices of the clot using their antibodies8. The microscopic image-based methods are only concerned with the specific structure of the clot9. Moreover, all those methods are laborious and time-consuming10. To date, the procedures for quantitatively and qualitatively studying cerebral thrombi cell components have not been reported. It is widely acknowledged that the cross-linked fibrin tightly wraps the blood cells in the clots11. Consequently, the specific degradation of the cross-linked fibrin and release of the intact cells is critical for the accurate analysis of cell components.
Previous works isolated a fibrinolytic enzyme from Sipunculus nudus (sFE), which can degrade the fibrin specifically and quickly12. Herein, a method for analyzing the cell components of the cerebral thrombi based on the unique activity of sFE was proposed. This protocol utilized sFE to degrade the fibrin of clots first and then analyzed the cell components by Wright&#39;s Staining and routine blood examination13,14. According to this method, the cell components of cerebral thrombi can be quantitatively and qualitatively studied. This simple and effective protocol might be applied for the cell component analysis of other blood clots.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agglutination Reaction Plate</td>
			<td>ROTEST</td>
			<td>RTB-4003</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto Hematology Analyzer</td>
			<td>SYSMEX</td>
			<td>XNB2</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic Vertical Pressure Steam Sterilizer&#160;</td>
			<td>SANYO</td>
			<td>MLS-3750</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube (1.5 mL)</td>
			<td>Biosharp</td>
			<td>BS-15-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>AIRTECH</td>
			<td>BLB-1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant Temperature Incubator</td>
			<td>JINGHONG</td>
			<td>JHS-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Dish (100 mm)</td>
			<td>NEST</td>
			<td>704001</td>
			<td></td>
		</tr>
		<tr>
			<td>DHG Series Heating and Drying Oven&#160;</td>
			<td>SENXIN</td>
			<td>DGG-9140AD</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Analytical Balance</td>
			<td>DENVER</td>
			<td>TP-213</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Membrane (0.22 &#181;m)</td>
			<td>Millex GP</td>
			<td>SLGP033NK</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Refrigerated Centrifuge&#160;</td>
			<td>Cence</td>
			<td>H1650-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>CITOGLAS</td>
			<td>01-30253-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q Reference</td>
			<td>Millipore</td>
			<td>Z00QSV0CN</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>CISEN</td>
			<td>H37022337</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE E100</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>Beyotime</td>
			<td>C0221A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (1 mL )</td>
			<td>Axygene</td>
			<td>T-1000XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (200 &#181;L)</td>
			<td>Axygene</td>
			<td>T-200XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (1 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY18723</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (200 &#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY20280</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>MARTOR</td>
			<td>23111</td>
			<td></td>
		</tr>
		<tr>
			<td>Small-sized Vortex Oscillator</td>
			<td>Kylin-Bell</td>
			<td>VORTEX KB3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Hystic</td>
			<td>HKQS-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Wright Staining Solution</td>
			<td>Beyotime</td>
			<td>C0135-500ml</td>
			<td></td>
		</tr>
	</tbody>
</table>

a comprehensive approach to analyze the cell components of cerebral blood clots

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

Author Spotlight: A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots 

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

In the initial stage of the degradation process, it was found that the blood clots had a red compact structure, and the working solution was colorless. After incubation for 30 min, the working solution turned light red, which indicated the crossed blood cells were released into the working solution. Most clots were dissolved when lengthening the incubation time to 5 h, and the working solution became light red. On the contrary, there was no significant change in the physiological saline group (NC) even after 10 h incubat...

Watch this Scientific Journal Video about A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots at JoVE.com

This study describes a fast and effective method for the cell component analysis of cerebral blood clots through clot dissolving, cell staining, and routine blood examination.

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...

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456 Views.  Quanzhou First Hospital Affiliated to Fujian Medical University. This study describes a fast and effective method for the cell component analysis of cerebral blood clots through clot dissolving, cell staining, and routine blood examination.