Name: Autore in primo piano: Un nuovo metodo per l'analisi completa delle componenti cellulari dei coaguli di sangue cerebrale
Uploaded: 2023-07-21T13:30:00
Description: Watch this Scientific Journal Video about A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots at JoVE.com

Questa ricerca è stata finanziata dall'Ufficio per la scienza e la tecnologia della città di Xiamen (3502Z20227197) e dall'Ufficio per la scienza e la tecnologia della provincia del Fujian (n. 2019J01070, n. 2021Y0027).

La ricerca è stata condotta in conformità con le linee guida istituzionali del Comitato di Etica Medica dell'Università di Huaqiao. I coaguli di sangue cerebrale sono stati rimossi chirurgicamente e raccolti presso il Quanzhou First Hospital, affiliato alla Fujian Medical University, con il consenso informato dei pazienti.
1. Pretrattamento del coagulo di sangue
<ol><li>Disporre i coaguli su un piatto pulito, aggiungere 5 ml di soluzione fisiologica con una pinzetta, agit...

Un metodo basato su enzimi fibrinolitici per studiare i componenti delle cellule del trombo cerebrale

<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. 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sFE è un agente fibrinolitico in grado di degradare la fibrina direttamente ed efficacemente12,16. Qui, sFE è stato impiegato per degradare la fibrina reticolata dei coaguli di sangue cerebrale, rilasciare le cellule racchiuse all'interno dei coaguli e analizzare qualitativamente e quantitativamente i componenti cellulari dei coaguli. I dati della microscopia e l'esame del sangue di routine hanno indicato che le cellule racchiuse sono state rilasciate dai coagu...

La malattia cerebrovascolare è una delle tre principali malattie che possono minacciare la salute umana, tra le quali la malattia cerebrovascolare ischemica rappresenta oltre l'80%. La trombosi cerebrale e la trombosi venosa cerebrale sono oggi le malattie cerebrovascolari ischemiche più preoccupanti, causate principalmente da coaguli di sangue cerebrale 1,2. Se il trattamento non viene eseguito correttamente, avrà alti tassi di disabilità e mortalità e un alto tasso di recidiva dopo la dimissione3.
Recentemente, un numero crescente di studi ha dimostrato che i componenti cellulari dei coaguli di sangue cerebrale sono strettamente correlati con la diagnosi, il trattamento e la prognosi della trombosi cerebrale 4,5,6. Pertanto, la disponibilità di dati sulla composizione del trombo, in particolare sui componenti cellulari, è importante per la diagnosi clinica e il trattamento. Sfortunatamente, i metodi attualmente disponibili non sono in grado di analizzare in modo completo la componente del coagulo di sangue quantitativamente e qualitativamente. Ad esempio, la colorazione in situ a base di Martius Scarlett Blue può studiare solo i globuli rossi/bianchi di alcune fette del coagulo7. La colorazione in situ basata sull'immunoistochimica (IHC) può studiare solo i componenti limitati del sangue di alcune fette del coagulo utilizzando i loro anticorpi8. I metodi basati su immagini microscopiche riguardano solo la struttura specifica del coagulo9. Inoltre, tutti questi metodi sono laboriosi e richiedono molto tempo10. Ad oggi, le procedure per lo studio quantitativo e qualitativo dei componenti delle cellule trombiformi cerebrali non sono state riportate. È ampiamente riconosciuto che la fibrina reticolata avvolge strettamente le cellule del sangue nei coaguli11. Di conseguenza, la degradazione specifica della fibrina reticolata e il rilascio delle cellule intatte sono fondamentali per l'analisi accurata dei componenti cellulari.
Lavori precedenti hanno isolato un enzima fibrinolitico da Sipunculus nudus (sFE), che può degradare la fibrina in modo specifico e rapido12. In questo caso, è stato proposto un metodo per analizzare i componenti cellulari dei trombi cerebrali basato sull'attività unica di sFE. Questo protocollo ha utilizzato sFE per degradare prima la fibrina dei coaguli e poi ha analizzato i componenti cellulari mediante colorazione di Wright ed esame del sanguedi routine 13,14. Secondo questo metodo, i componenti cellulari dei trombi cerebrali possono essere studiati quantitativamente e qualitativamente. Questo protocollo semplice ed efficace potrebbe essere applicato per l'analisi della componente cellulare di altri coaguli di sangue.

<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

La trombosi cerebrale, un coagulo di sangue in un'arteria o vena cerebrale, è il tipo più comune di infarto cerebrale. Lo studio dei componenti cellulari dei coaguli di sangue cerebrale è importante per la diagnosi, il trattamento e la prognosi. Tuttavia, gli attuali approcci allo studio dei componenti cellulari dei coaguli si basano principalmente sulla colorazione in situ , che non è adatta per lo studio completo dei componenti cellulari perché le cellule sono strettamente avvolte nei coaguli. Studi precedenti hanno isolato con successo un enzima fibrinolitico (sFE) da Sipunculus nudus, che può degradare direttamente la fibrina reticolata, rilasciando i componenti cellulari. Questo studio ha stabilito un metodo completo basato sull'sFE per studiare i componenti cellulari del trombo cerebrale. Questo protocollo include la dissoluzione del coagulo, il rilascio cellulare, la colorazione cellulare e l'esame del sangue di routine. Secondo questo metodo, i componenti cellulari potrebbero essere studiati quantitativamente e qualitativamente. Vengono mostrati i risultati rappresentativi degli esperimenti che utilizzano questo metodo.

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.

Autore in primo piano: Un nuovo metodo per l'analisi completa delle componenti cellulari dei coaguli di sangue cerebrale 

Un approccio completo per analizzare i componenti cellulari dei coaguli di sangue cerebrale

Our research mainly focuses on cerebral thrombosis. Many studies have shown that cerebral blood clot cell component correlate with the diagnosis, treatment, and prognosis of cerebral thrombosis. In this study, we established a novel method for the analysis of cell component of cerebral blood clots.This challenge in analyzing cell components of cerebral blood clots is the release of entire shaft from blood clots. This is because the clots leaked fibering tiry rash, the blood cells in the clots. Targeted approaches to starting the cell components of the clots are best done in C2 standing, which is unsuitable for the comprehensive starting of the cell components.On the contrary, our method enables both quantitative and qualitative enlightenings of the cerebral slope cell components. Our method utilizes fibrinolytic enzyme to delay the fibering in blood clots, releasing the intact cells. The data obtained from the analysis of the cell components will facilitate the study of the mechanisms of cerebral thrombosis at a molecular level.Our team will continue to focus on the study of fibrinolysis and coagulation system.

Nella fase iniziale del processo di degradazione, è stato riscontrato che i coaguli di sangue avevano una struttura compatta rossa e la soluzione di lavoro era incolore. Dopo un'incubazione di 30 minuti, la soluzione di lavoro è diventata di colore rosso chiaro, il che indica che le cellule del sangue incrociate sono state rilasciate nella soluzione di lavoro. La maggior parte dei coaguli si è dissolta quando si è allungato il tempo di incubazione a 5 ore e la soluzione di lavoro è diventata rosso chiaro. Al contrar...

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

Questo studio descrive un metodo rapido ed efficace per l'analisi dei componenti cellulari dei coaguli di sangue cerebrale attraverso la dissoluzione del coagulo, la colorazione cellulare e l'esame del sangue di routine.

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 ...

La nostra ricerca si concentra principalmente sulla trombosi cerebrale. Molti studi hanno dimostrato che la componente delle cellule del coagulo di sangue cerebrale è correlata alla diagnosi, al trattamento e alla prognosi della trombosi cerebrale. In questo studio, abbiamo stabilito un nuovo metodo per l'analisi della componente cellulare dei coaguli di sangue cerebrale.Questa sfida nell'analisi dei componenti cellulari dei coaguli di sangue cerebrale è il rilascio dell'intero albero dai coaguli di sangue. Questo perché i coaguli hanno perso fibrosi, eruzione cutanea, le cellule del sangue nei coaguli. Gli approcci mirati per avviare i componenti cellulari dei coaguli sono meglio eseguiti in posizione C2, che non è adatta per l'avvio completo dei componenti cellulari.Al contrario, il nostro metodo consente di illuminare sia quantitativamente che qualitativamente le componenti delle cellule di pendenza cerebrale. Il nostro metodo utilizza l'enzima fibrinolitico per ritardare la fibratura nei coaguli di sangue, rilasciando le cellule intatte. I dati ottenuti dall'analisi dei componenti cellulari faciliteranno lo studio dei meccanismi della trombosi cerebrale a livello molecolare.Il nostro team continuerà a concentrarsi sullo studio della fibrinolisi e del sistema di coagulazione.

a-comprehensive-approach-to-analyze-cell-components-cerebral-blood

Research

JoVE Journal

Biochemistry

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.

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.

Ricerca

Biochimica

Cerebral Thrombus Clot Dissolution and Cell Staining for Cell Component Analyses

To begin, place the collected cerebral blood clot samples on a clean dish using tweezers. Then, using a pipette, add five milliliters of physiological saline to it and gently shake the dish. Remove the saline using a pipette.Using a pair of scissors, chop the clots into small pieces. Once again, add five milliliters of physiological saline to the clots and shake the dish gently before aspirating the saline with a pipette. Then, using tweezers, transfer the pieces of clots to a new, clean U-plate.Prepare the SFE working solution by adjusting the concentration of SFE to 2, 000 units per milliliter using physiological saline. Add 300 microliters of the SFE working solution to the pretreated clots. To initiate the first round of degradation, incubate the mixture of SFE and clots at 37 degrees Celsius for half an hour.After that, gently shake to mix the SFE treated sample. And using a clean pipette, transfer the liquid portion to a sterile tube. Set aside the remaining clot for another round of degradation.Centrifuge the collected liquid at 200 G for five minutes at four degrees Celsius. Next, using a pipette, transfer the supernatant or the recovered SFE solution to another clean tube. Resuspend the resultant cell pellet in 50 microliters of physiological saline by gentle mixing.After this, perform subsequent rounds of degradation on the remaining clots using the recovered SFE solution as demonstrated previously. Gather the cell mixture from each round of degradation to prepare the blood cell sample. Add five microliters of the obtained cell sample to the poly-L-lysine-coated glass slide.And using a cover slip, smear the cells. Allow the liquid to evaporate at room temperature. To perform cell staining, gently add 100 microliters of rights dye to the cell smear.Allow the cells to stain for 30 minutes at room temperature before gently rinsing the dye off with clean water. Finally, examine the stained cells using a light microscope. Compact red blood clots with a colorless working solution were observed during the early stage of degradation.Clot dissolution was observed after a 30-minute incubation and a prolonged incubation of up to five hours dissolved most clots, while there was no significant change in the negative control group, even after a 10-hour incubation. After a right staining, mature red blood cells, platelets, and various granulocytes were clearly identifiable under a light microscope. The cell components could be quantitatively analyzed with the aid of autohematology successfully.