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

Summary

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.

Abstract

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.

Introduction

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 clots^1,2. 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 thrombosis^4,5,6. 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's Staining and routine blood examination^13,14. 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.

Protocol

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 pipette.
   NOTE: Repeat the rinse three times.
2. Cut the clots into smaller pieces (smaller than 5 mm x 5 mm x 5 mm) with a scissor. Add 5 mL of physiological saline, shake the dish gently, and remove the saline with a pipette.
   NOTE: Repeat the rinse three times. Ensure no clot pieces are aspirated during the rinse.
3. Transfer the clots to another clean U-plate.
   ​NOTE: The protocol can be paused here (store the plate at 2-8 °C) and restarted later.

2. Thrombolysis

1. Prepare the sFE working solution by adjusting the concentration of sFE to 2000 U/mL with physiological saline.
   NOTE: The sFE was prepared according to the well-established protocols of Tang Lab¹⁵.
2. Add 300 µL of sFE working solution into the pretreated clots.
3. Perform the first round of degradation.
   1. Incubate the mixture of sFE and clots at 37 °C for 0.5 h.
      NOTE: The blot treated with physiological saline was set as negative control. Do not rotate the sample on the shaker.
   2. Mix the sample gently. Transfer the liquid part to another clean tube with a clean pipette.
      NOTE: The remaining clots were used for another round of degradation.
   3. Centrifuge the liquid part at 200 × g for 5 min at 4 °C.
   4. Transfer the supernatant to another clean tube with a pipette to obtain the recovered sFE solution.
   5. Mix the cell precipitation with 50 µL of physiological saline gently.
      NOTE: The cell mixture was stored 2-8 °C for later use.
4. Perform subsequent degradation.
   1. Degrade the remaining clots (step 2.3.2) with the recovered sFE solution (step 2.3.4) at 37 °C for 0.5 h.
      NOTE: The rest steps were the same as the first degradation round. The degradation process was finished until the whole clots were degraded.
5. Perform cell collection.
   1. Prepare the blood cell sample by collecting the cell mixture of each round of degradation.

3. Wright's staining

1. Perform cell smear.
   1. Adjust cells to a density of 1 x 10⁶ cells/mL.
   2. Add 5 µL of the cells to the poly-L lysine-coated glass slide, then smear them using the cover slip.
   3. Evaporate the liquid at room temperature.
2. Perform the staining.
   1. Add 200 µL of Wright's dye (see Table of Materials) to the cell smear gently, and add 600 µL of Wright's dye B to mix evenly.  Stain for 10 min at room temperature.
   2. Rinse the dye gently with clean water.
      NOTE: The stained cell smear can be stored at room temperature for later use.
3. Perform microscopic imaging.
   1. Examine the stained cells using a light microscope (see Table of Materials).

4. Routine blood examination

1. Adjust cells to a density of 1 x 10⁶ cells/mL.
2. Analyze the cell components such as platelets, erythrocytes, white blood cells, lymphocytes, neutrophils, monocytes, eosinophils, basophils, and immature granulocytes using an autohematology analyzer according to the manufacturer's instructions (see Table of Materials).

Results

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...

Discussion

sFE is a fibrinolytic agent that can degrade the fibrin directly and effectively^12,16. 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...

Materials

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