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Abstract

Introduction

Protocol

Representative Results

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Materials

References

Biochemistry

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

Published: July 21st, 2023

DOI:

10.3791/65791

1Emergency Department, Quanzhou First Hospital Affiliated to Fujian Medical University, 2Medicine School, Huaqiao University, 3Department of Haematology, Quanzhou First Hospital Affiliated to Fujian Medical University, 4Department of Laboratory Medicine, Quanzhou First Hospital Affiliated to Fujian Medical University
* These authors contributed equally

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

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

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 correla....

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The research was performed in compliance with the institutional guidelines of the Medical Ethics Committee of Huaqiao University. The cerebral blood clots were surgically removed and collected at Quanzhou First Hospital, affiliated to Fujian Medical University, with informed consent from the patients.

1. Blood clot pretreatment

  1. Place the clots on a clean dish, add 5 mL of physiological saline with a tweezer, shake the dish gently, and remove the saline with a pipet.......

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

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

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

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

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