Name: 저자 스포트라이트: 뇌혈전의 포괄적인 세포 구성 성분 분석을 위한 새로운 방법
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

이 연구는 샤먼시 과학기술국(3502Z20227197)과 푸젠성 과학기술국(No. 2019J01070, No.2021Y0027)의 지원을 받았습니다.

이 연구는 Huaqiao University 의료 윤리 위원회의 기관 지침에 따라 수행되었습니다. 뇌혈전은 푸젠 의과대학 부속 취안저우 제1병원에서 환자의 사전 동의를 얻어 외과적으로 제거해 채취했다.
1. 혈전 전처리
<ol><li>깨끗한 접시에 혈전을 놓고 핀셋으로 생리식염수 5mL를 넣고 접시를 부드럽게 흔든 다음 피펫으로 식염수를 제거합니다. 알림: 헹굼을 세 번 반복합?...

대뇌 혈전 세포 구성 요소를 연구하기 위한 섬유소 용해 효소 기반 방법

<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>

sFE는 피브린을 직접적이고 효과적으로 분해할 수 있는 섬유소 용해제입니다12,16. 여기서 sFE를 사용하여 대뇌 혈전의 가교 피브린을 분해하고, 혈전 내에 밀폐된 세포를 방출하고, 혈전의 세포 성분을 정성적, 정량적으로 분석했습니다. 현미경 검사 데이터와 정기 혈액 검사는 밀폐된 세포가 혈전에서 방출되었음을 나타냈습니다. 또한, 방출된 세포의 ?...

뇌혈관질환은 인간의 건강을 위협할 수 있는 3대 질환 중 하나로, 그 중 허혈성 뇌혈관질환이 80% 이상을 차지합니다. 뇌혈전증과 뇌정맥혈전증은 오늘날 가장 우려되는 허혈성 뇌혈관 질환으로, 주로 뇌혈전으로 인해 발생한다 1,2. 치료가 제대로 이루어지지 않으면 장애율과 사망률이 높고 퇴원 후 재발률이 높다3.
최근에는 뇌혈전의 세포 성분이 뇌혈전증의 진단, 치료 및 예후와 밀접한 상관관계가 있다는 연구 결과가 점점 더 많이 나오고 있다 4,5,6. 따라서 혈전 구성, 특히 세포 구성 요소에 대한 데이터의 가용성은 임상 진단 및 치료에 중요합니다. 안타깝게도, 현재 사용 가능한 방법으로는 혈전 성분을 정량적, 정성적으로 종합적으로 분석할 수 없습니다. 예를 들어, Martius Scarlett Blue 기반 in-situ 염색은 혈전7의 특정 절편의 적혈구/백혈구만 연구할 수 있습니다. 면역조직화학(IHC) 기반 in-situ 염색은 항체를 사용하여 혈전의 특정 절편의 제한된 혈액 성분만 연구할 수 있다8. 현미경적 이미지 기반 방법은 응고(9)의 특정 구조에만 관련된다. 더욱이 이러한 모든 방법은 힘들고 시간이 많이 걸립니다10. 현재까지 대뇌혈전세포 성분을 정량적, 정성적으로 연구하는 절차는 보고되지 않았다. 가교된 피브린이 혈전의 혈액 세포를 단단히 감싸는 것은 널리 알려져있다 11. 결과적으로, 가교된 피브린의 특이적 분해와 온전한 세포의 방출은 세포 성분의 정확한 분석에 매우 중요합니다.
이전 연구에서는 피브린을 특이적이고 빠르게 분해할 수 있는 Sipunculus nudus(sFE)에서 섬유소 용해 효소를 분리했습니다12. 본 연구에서는 sFE의 독특한 활성에 기초하여 뇌혈전의 세포 성분을 분석하는 방법을 제안하였다. 이 프로토콜은 sFE를 사용하여 먼저 혈전의 피브린을 분해한 다음 Wright의 염색 및 정기 혈액 검사13,14로 세포 성분을 분석했습니다. 이 방법에 따르면 뇌혈전의 세포 성분을 정량적, 정성적으로 연구할 수 있다. 이 간단하고 효과적인 프로토콜은 다른 혈전의 세포 성분 분석에 적용될 수 있습니다.

<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

뇌동맥이나 정맥에 혈전이 생기는 뇌혈전증은 뇌경색의 가장 흔한 유형이다. 뇌혈전의 세포 성분에 대한 연구는 진단, 치료 및 예후에 중요합니다. 그러나 혈전의 세포 성분을 연구하는 현재의 접근 방식은 주로 in situ 염색을 기반으로 하며, 이는 세포가 혈전으로 단단히 싸여 있기 때문에 세포 성분에 대한 포괄적인 연구에는 적합하지 않습니다. 이전 연구에서는 Sipunculus nudus에서 섬유소 용해 효소(sFE)를 성공적으로 분리했는데, 이는 가교 결합된 피브린을 직접 분해하여 세포 성분을 방출할 수 있습니다. 이 연구는 뇌혈전의 세포 성분을 연구하기 위해 sFE를 기반으로 하는 포괄적인 방법을 확립했습니다. 이 프로토콜에는 혈전 용해, 세포 방출, 세포 염색 및 정기 혈액 검사가 포함됩니다. 이 방법에 따르면 세포 구성 요소를 정량적 및 정성적으로 연구할 수 있습니다. 이 방법을 사용한 실험의 대표적인 결과를 나타낸다.

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.

저자 스포트라이트: 뇌혈전의 포괄적인 세포 구성 성분 분석을 위한 새로운 방법 

뇌혈전의 세포 성분을 분석하기 위한 포괄적인 접근법

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.

분해 과정의 초기 단계에서 혈전은 적색 조밀 구조를 가지고 있으며 작업 용액은 무색이라는 것이 밝혀졌습니다. 30분 동안 배양한 후, 작업 용액은 밝은 빨간색으로 변했으며, 이는 교차된 혈액 세포가 작업 용액으로 방출되었음을 나타냅니다. 배양 시간을 5시간으로 늘리면 대부분의 혈전이 용해되었고, 작업 용액은 연한 붉은색이 되었습니다. 반대로, 10시간 배양 후에도 생리식염수군(NC)에는 ?...

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

본 연구는 혈전 용해, 세포 염색 및 정기 혈액 검사를 통한 뇌혈전의 세포 성분 분석을 위한 빠르고 효과적인 방법을 설명합니다.

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 ...

우리의 연구는 주로 뇌혈전증에 초점을 맞추고 있습니다. 많은 연구에서 뇌혈전 세포 성분이 뇌혈전증의 진단, 치료 및 예후와 상관관계가 있음을 보여주었습니다. 이 연구에서는 뇌혈전의 세포 성분을 분석하기 위한 새로운 방법을 확립했습니다.뇌 혈전의 세포 성분을 분석하는 데 있어 이 과제는 혈전에서 전체 샤프트를 방출하는 것입니다. 혈전이 혈전 속의 혈액 세포인 섬유질 발진을 누출했기 때문이다. 혈전의 세포 성분을 시작하는 표적 접근법은 세포 성분의 포괄적인 시작에 적합하지 않은 C2 스탠딩에서 가장 잘 수행됩니다.반대로, 우리의 방법은 대뇌 경사 세포 구성 요소의 양적 및 질적 계몽을 모두 가능하게 합니다. 우리의 방법은 섬유소 용해 효소를 사용하여 혈전의 섬유화를 지연시켜 온전한 세포를 방출합니다. 세포 성분 분석에서 얻은 데이터는 분자 수준에서 뇌 혈전증의 메커니즘 연구를 용이하게 할 것입니다.우리 팀은 섬유소 용해 및 응고 시스템 연구에 계속 집중할 것입니다.

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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.

연구

생화학

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.