Name: Forfatter Spotlight: En ny metode til omfattende cellekomponentanalyse af cerebrale blodpropper
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

Denne forskning blev finansieret af Science and Technology Bureau of Xiamen City (3502Z20227197) og Science and Technology Bureau of Fujian-provinsen (nr. 2019J01070, nr. 2021Y0027).

Forskningen blev udført i overensstemmelse med de institutionelle retningslinjer fra den medicinske etiske komité ved Huaqiao University. De cerebrale blodpropper blev fjernet kirurgisk og indsamlet på Quanzhou First Hospital, tilknyttet Fujian Medical University, med informeret samtykke fra patienterne.
1. Forbehandling af blodpropper
<ol><li>Placer blodpropperne på en ren skål, tilsæt 5 ml fysiologisk saltvand med en pincet, ryst skålen forsigtigt og fjern saltvand ...

En fibrinolytisk enzymbaseret metode til at studere cerebrale trombecellekomponenter

<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>

sFE er et fibrinolytisk middel, der kan nedbryde fibrinet direkte og effektivt12,16. Her blev sFE anvendt til at nedbryde den tværbundne fibrin i cerebrale blodpropper, frigive de lukkede celler i blodpropperne og analysere blodproppernes cellekomponenter kvalitativt og kvantitativt. Mikroskopidataene og rutinemæssig blodprøve indikerede, at de lukkede celler blev frigivet fra blodpropperne. Desuden blev celletyperne og strukturerne i de frigivne celler ikke p...

Cerebrovaskulær sygdom er en af tre store sygdomme, der kan true menneskers sundhed, blandt hvilke iskæmisk cerebrovaskulær sygdom tegner sig for mere end 80%. Cerebral trombose og cerebral venetrombose er de mest bekymrede iskæmiske cerebrovaskulære sygdomme i dag, hovedsageligt forårsaget af cerebrale blodpropper 1,2. Hvis behandlingen ikke udføres korrekt, vil den have høje invaliditets- og dødelighedsrater og en høj tilbagefaldsrate efter udskrivning3.
For nylig har et stigende antal undersøgelser vist, at cellekomponenterne i cerebrale blodpropper er tæt korreleret med diagnosen, behandlingen og prognosen for cerebral trombose 4,5,6. Derfor er tilgængeligheden af data om trombosesammensætning, især cellekomponenterne, vigtig for klinisk diagnose og behandling. Desværre kan de aktuelt tilgængelige metoder ikke omfattende analysere blodpropkomponenten kvantitativt og kvalitativt. For eksempel kan Martius Scarlett Blue-baseret in situ-farvning kun studere de røde / hvide blodlegemer i visse skiver afblodproppen 7. Immunohistokemi (IHC) baseret in situ farvning kan kun studere begrænsede blodkomponenter i visse skiver af blodproppen ved hjælp af deres antistoffer8. De mikroskopiske billedbaserede metoder beskæftiger sig kun med den specifikke struktur afblodproppen 9. Desuden er alle disse metoder besværlige og tidskrævende10. Hidtil er procedurerne for kvantitativ og kvalitativ undersøgelse af cerebrale trombicellekomponenter ikke blevet rapporteret. Det er almindeligt anerkendt, at det tværbundne fibrin tæt omslutter blodcellerne i blodpropperne11. Derfor er den specifikke nedbrydning af det tværbundne fibrin og frigivelsen af de intakte celler afgørende for den nøjagtige analyse af cellekomponenter.
Tidligere værker isolerede et fibrinolytisk enzym fra Sipunculus nudus (sFE), som kan nedbryde fibrinet specifikt og hurtigt12. Heri blev der foreslået en metode til analyse af cellekomponenterne i cerebral trombi baseret på den unikke aktivitet af sFE. Denne protokol brugte sFE til først at nedbryde fibrin af blodpropper og analyserede derefter cellekomponenterne ved Wrights farvning og rutinemæssige blodundersøgelse13,14. Ifølge denne metode kan cellekomponenterne i cerebral thrombi undersøges kvantitativt og kvalitativt. Denne enkle og effektive protokol kan anvendes til cellekomponentanalyse af andre blodpropper.

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			<td>Agglutination Reaction Plate</td>
			<td>ROTEST</td>
			<td>RTB-4003</td>
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			<td>Auto Hematology Analyzer</td>
			<td>SYSMEX</td>
			<td>XNB2</td>
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			<td>Automatic Vertical Pressure Steam Sterilizer&#160;</td>
			<td>SANYO</td>
			<td>MLS-3750</td>
			<td></td>
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			<td>Centrifuge Tube (1.5 mL)</td>
			<td>Biosharp</td>
			<td>BS-15-M</td>
			<td></td>
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			<td>Clean bench</td>
			<td>AIRTECH</td>
			<td>BLB-1600</td>
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			<td>Constant Temperature Incubator</td>
			<td>JINGHONG</td>
			<td>JHS-400</td>
			<td></td>
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			<td>Culture Dish (100 mm)</td>
			<td>NEST</td>
			<td>704001</td>
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			<td>DHG Series Heating and Drying Oven&#160;</td>
			<td>SENXIN</td>
			<td>DGG-9140AD</td>
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			<td>Electronic Analytical Balance</td>
			<td>DENVER</td>
			<td>TP-213</td>
			<td></td>
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			<td>Filter Membrane (0.22 &#181;m)</td>
			<td>Millex GP</td>
			<td>SLGP033NK</td>
			<td></td>
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			<td>Micro Refrigerated Centrifuge&#160;</td>
			<td>Cence</td>
			<td>H1650-W</td>
			<td></td>
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		<tr>
			<td>Microscope Slides</td>
			<td>CITOGLAS</td>
			<td>01-30253-50</td>
			<td></td>
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			<td>Milli-Q Reference</td>
			<td>Millipore</td>
			<td>Z00QSV0CN</td>
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			<td>Normal Saline</td>
			<td>CISEN</td>
			<td>H37022337</td>
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			<td>Optical Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE E100</td>
			<td></td>
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			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
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			<td>Phosphate-Buffered Saline</td>
			<td>Beyotime</td>
			<td>C0221A</td>
			<td></td>
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			<td>Pipette Tip (1 mL )</td>
			<td>Axygene</td>
			<td>T-1000XT-C</td>
			<td></td>
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			<td>Pipette Tip (200 &#181;L)</td>
			<td>Axygene</td>
			<td>T-200XT-C</td>
			<td></td>
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			<td>Pipettor (1 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY18723</td>
			<td></td>
		</tr>
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			<td>Pipettor (200 &#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY20280</td>
			<td></td>
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			<td>Scalpel</td>
			<td>MARTOR</td>
			<td>23111</td>
			<td></td>
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			<td>Small-sized Vortex Oscillator</td>
			<td>Kylin-Bell</td>
			<td>VORTEX KB3</td>
			<td></td>
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			<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>
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a comprehensive approach to analyze the cell components of cerebral blood clots

Cerebral trombose, en blodprop i en cerebral arterie eller vene, er den mest almindelige type cerebral infarkt. Undersøgelsen af cellekomponenterne i cerebrale blodpropper er vigtig for diagnose, behandling og prognose. Imidlertid er de nuværende tilgange til at studere blodproppernes cellekomponenter hovedsageligt baseret på in situ-farvning , hvilket er uegnet til den omfattende undersøgelse af cellekomponenterne, fordi cellerne er tæt pakket ind i blodpropperne. Tidligere undersøgelser har med succes isoleret et fibrinolytisk enzym (sFE) fra Sipunculus nudus, som kan nedbryde det tværbundne fibrin direkte og frigive cellekomponenterne. Denne undersøgelse etablerede en omfattende metode baseret på sFE til at studere cellekomponenterne i cerebral trombose. Denne protokol omfatter koagulationsopløsning, cellefrigivelse, cellefarvning og rutinemæssig blodundersøgelse. Ifølge denne metode kunne cellekomponenterne undersøges kvantitativt og kvalitativt. De repræsentative resultater af eksperimenter ved hjælp af denne metode vises.

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.

Forfatter Spotlight: En ny metode til omfattende cellekomponentanalyse af cerebrale blodpropper 

En omfattende tilgang til analyse af cellekomponenterne i cerebrale blodpropper

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.

I den indledende fase af nedbrydningsprocessen blev det konstateret, at blodpropperne havde en rød kompakt struktur, og arbejdsløsningen var farveløs. Efter inkubation i 30 minutter blev arbejdsløsningen lys rød, hvilket indikerede, at de krydsede blodlegemer blev frigivet til arbejdsløsningen. De fleste blodpropper blev opløst ved forlængelse af inkubationstiden til 5 timer, og arbejdsløsningen blev lys rød. Tværtimod var der ingen signifikant ændring i den fysiologiske saltvandsgruppe (NC) selv efter 10 tim...

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

Denne undersøgelse beskriver en hurtig og effektiv metode til cellekomponentanalyse af cerebrale blodpropper gennem koagulationsopløsning, cellefarvning og rutinemæssig blodundersøgelse.

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 ...

Vores forskning fokuserer primært på blodpropper i hjernen. Mange undersøgelser har vist, at cerebral blodpropcellekomponent korrelerer med diagnose, behandling og prognose for cerebral trombose. I denne undersøgelse etablerede vi en ny metode til analyse af cellekomponent i cerebrale blodpropper.Denne udfordring ved analyse af cellekomponenter i cerebrale blodpropper er frigivelsen af hele skaftet fra blodpropper. Dette skyldes, at blodpropperne lækkede fibering tiry udslæt, blodcellerne i blodpropperne. Målrettede tilgange til at starte cellekomponenterne i blodpropperne udføres bedst i C2-stående, hvilket er uegnet til omfattende start af cellekomponenterne.Tværtimod muliggør vores metode både kvantitativ og kvalitativ oplysning af cellekomponenterne i hjerneskråningen. Vores metode bruger fibrinolytisk enzym til at forsinke fiberdannelsen i blodpropper og frigive de intakte celler. De data, der opnås ved analysen af cellekomponenterne, vil lette undersøgelsen af mekanismerne for cerebral trombose på molekylært niveau.Vores team vil fortsat fokusere på studiet af fibrinolyse og koagulationssystem.

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.

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.