Name: Författare i fokus: En ny metod för omfattande cellkomponentanalys av cerebrala blodproppar
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

Denna forskning finansierades av Science and Technology Bureau of Xiamen City (3502Z20227197) och Science and Technology Bureau of Fujian Province (No. 2019J01070, No.2021Y0027).

Forskningen utfördes i enlighet med de institutionella riktlinjerna från den medicinska etikkommittén vid Huaqiao University. De cerebrala blodpropparna opererades bort och samlades in på Quanzhou First Hospital, som är anslutet till Fujian Medical University, med informerat samtycke från patienterna.
1. Förbehandling av blodproppar
<ol><li>Lägg blodpropparna på en ren tallrik, tillsätt 5 ml fysiologisk koksaltlösning med en pincett, skaka skålen försiktigt och ...

En fibrinolytisk enzymbaserad metod för att studera cerebrala trombocytkomponenter

<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 är ett fibrinolytiskt medel som kan bryta ned fibrinet direkt och effektivt12,16. Här användes sFE för att bryta ned det tvärbundna fibrinet i hjärnblodpropparna, frigöra de inneslutna cellerna i koapelarna och analysera cellkomponenterna i blodpropparna kvalitativt och kvantitativt. Mikroskopidata och rutinmässig blodundersökning indikerade att de inneslutna cellerna frigjordes från blodpropparna. Celltyperna och strukturerna hos de frisatta celler...

Cerebrovaskulär sjukdom är en av tre stora sjukdomar som kan hota människors hälsa, bland vilka ischemisk cerebrovaskulär sjukdom står för mer än 80%. Cerebral trombos och cerebral ventrombos är de mest drabbade ischemiska cerebrovaskulära sjukdomarna idag, främst orsakade av cerebrala blodproppar 1,2. Om behandlingen inte görs på rätt sätt kommer den att ha höga funktionshinder och dödlighet och en hög återfallsfrekvens efter utskrivning3.
På senare tid har ett växande antal studier visat att cellkomponenterna i hjärnans blodproppar är nära korrelerade med diagnos, behandling och prognos av cerebral trombos 4,5,6. Därför är tillgången till data om trombernas sammansättning, särskilt cellkomponenterna, viktig för klinisk diagnos och behandling. Tyvärr kan de metoder som för närvarande finns tillgängliga inte analysera blodproppskomponenten kvantitativt och kvalitativt. Till exempel kan Martius Scarlett Blue-baserad in-situ-färgning bara studera de röda/vita blodkropparna i vissa skivor av propp7. Immunohistokemi (IHC) baserad in-situ färgning kan endast studera begränsade blodkomponenter i vissa delar av blodproppen med hjälp av deras antikroppar8. De mikroskopiska bildbaserade metoderna är endast intresserade av den specifika strukturen hos propp9. Dessutom är alla dessa metoder mödosamma och tidskrävande10. Hittills har procedurerna för kvantitativ och kvalitativ studie av cerebrala trombicellskomponenter inte rapporterats. Det är allmänt erkänt att det tvärbundna fibrinet tätt lindar in blodkropparna i blodpropparna11. Följaktligen är den specifika nedbrytningen av det tvärbundna fibrinet och frisättningen av de intakta cellerna avgörande för en korrekt analys av cellkomponenterna.
Tidigare arbeten isolerade ett fibrinolytiskt enzym från Sipunculus nudus (sFE), som kan bryta ned fibrinet specifikt och snabbt12. Här föreslogs en metod för att analysera cellkomponenterna i cerebrala trombi baserat på den unika aktiviteten hos sFE. Detta protokoll använde sFE för att bryta ned fibrinet i blodproppar först och analyserade sedan cellkomponenterna genom Wright's Staining och rutinmässig blodundersökning13,14. Enligt denna metod kan cellkomponenterna i cerebrala tromber studeras kvantitativt och kvalitativt. Detta enkla och effektiva protokoll kan användas för analys av cellkomponenter i andra blodproppar.

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			<td>RTB-4003</td>
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			<td>Auto Hematology Analyzer</td>
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			<td>Automatic Vertical Pressure Steam Sterilizer&#160;</td>
			<td>SANYO</td>
			<td>MLS-3750</td>
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			<td>Centrifuge Tube (1.5 mL)</td>
			<td>Biosharp</td>
			<td>BS-15-M</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>
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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>DENVER</td>
			<td>TP-213</td>
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			<td>Filter Membrane (0.22 &#181;m)</td>
			<td>Millex GP</td>
			<td>SLGP033NK</td>
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			<td>Micro Refrigerated Centrifuge&#160;</td>
			<td>Cence</td>
			<td>H1650-W</td>
			<td></td>
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			<td>Microscope Slides</td>
			<td>CITOGLAS</td>
			<td>01-30253-50</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>
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			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
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			<td>Phosphate-Buffered Saline</td>
			<td>Beyotime</td>
			<td>C0221A</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>
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			<td>Pipettor (200 &#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY20280</td>
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			<td>Scalpel</td>
			<td>MARTOR</td>
			<td>23111</td>
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			<td>Small-sized Vortex Oscillator</td>
			<td>Kylin-Bell</td>
			<td>VORTEX KB3</td>
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			<td>Tweezer</td>
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			<td>Wright Staining Solution</td>
			<td>Beyotime</td>
			<td>C0135-500ml</td>
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a comprehensive approach to analyze the cell components of cerebral blood clots

Cerebral trombos, en blodpropp i en hjärnartär eller ven, är den vanligaste typen av hjärninfarkt. Studiet av cellkomponenterna i hjärnans blodproppar är viktigt för diagnos, behandling och prognos. De nuvarande metoderna för att studera cellkomponenterna i blodpropparna är dock huvudsakligen baserade på in situ-färgning , vilket är olämpligt för omfattande studier av cellkomponenterna eftersom cellerna är tätt inlindade i blodpropparna. Tidigare studier har framgångsrikt isolerat ett fibrinolytiskt enzym (sFE) från Sipunculus nudus, som kan bryta ner det tvärbundna fibrinet direkt och frigöra cellkomponenterna. Denna studie etablerade en omfattande metod baserad på sFE för att studera cellkomponenterna i cerebral trombos. Detta protokoll inkluderar proppupplösning, cellfrigöring, cellfärgning och rutinmässig blodundersökning. Enligt denna metod kunde cellkomponenterna studeras kvantitativt och kvalitativt. De representativa resultaten av experiment med denna metod visas.

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.

Författare i fokus: En ny metod för omfattande cellkomponentanalys av cerebrala blodproppar 

Ett omfattande tillvägagångssätt för att analysera cellkomponenterna i cerebrala blodproppar

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 det inledande skedet av nedbrytningsprocessen visade det sig att blodpropparna hade en röd kompakt struktur och att arbetslösningen var färglös. Efter inkubation i 30 minuter blev arbetslösningen ljusröd, vilket indikerade att de korsade blodkropparna frigjordes i arbetslösningen. De flesta blodproppar löstes upp när inkubationstiden förlängdes till 5 timmar, och arbetslösningen blev ljusröd. Tvärtom sågs ingen signifikant förändring i den fysiologiska koksaltgruppen (NC) även efter 10 timmars inkubat...

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

Denna studie beskriver en snabb och effektiv metod för cellkomponentanalys av cerebrala blodproppar genom proppupplösning, cellfärgning och rutinmässig blodundersökning.

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 ...

Vår forskning fokuserar främst på cerebral trombos. Många studier har visat att cerebrala blodproppscellkomponenter korrelerar med diagnos, behandling och prognos av cerebral trombos. I denna studie etablerade vi en ny metod för analys av cellkomponenten i cerebrala blodproppar.Denna utmaning vid analys av cellkomponenter i cerebrala blodproppar är frisättningen av hela skaftet från blodproppar. Detta beror på att propparna läckte ut fibertiryutslag, blodkropparna i blodpropparna. Riktade metoder för att starta cellkomponenterna i blodpropparna görs bäst i C2-stående, vilket är olämpligt för en omfattande start av cellkomponenterna.Tvärtom möjliggör vår metod både kvantitativ och kvalitativ upplysning av de cerebrala sluttande cellkomponenterna. Vår metod använder fibrinolytiskt enzym för att fördröja fiberbildningen i blodproppar och frigöra de intakta cellerna. De data som erhålls från analysen av cellkomponenterna kommer att underlätta studiet av mekanismerna för cerebral trombos på molekylär nivå.Vårt team kommer att fortsätta att fokusera på studier av fibrinolys och 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.