Large Volume Blood Collection from Swine for In Vitro Applications: Use of Intracardiac Cannulation and a Vacuum Pump as a Terminal Procedure

Summary

Here we present a method to collect a large volume of blood from swine using percutaneous intracardiac cannulation connected to a vacuum pump, as a terminal procedure.

Abstract

Routine blood collection is required in many basic and applied research studies. Multiple methods for swine blood collection are well described. These include using the auricular veins for the collection of small blood volumes, and the jugular veins or cranial vena cava for the collection of larger blood volumes. Some experiments require large volumes of blood from one individual animal at one time for analysis or use in primary cell cultures, which can best be achieved through exsanguination. Blood collected for primary cell culture must be freshly and carefully processed to maintain cellular integrity. We describe a method of aseptically harvesting blood from swine under a surgical plane of anesthesia, using a large-bore intravascular catheter stylet inserted percutaneously into the heart and a vacuum pump, which is efficient, provides a large yield, and minimizes cell lysis. This technique is performed once on an animal, at a surgical plane of anesthesia, as a terminal procedure, providing minimal risk for pain or distress.

Introduction

Pigs have been used in research in studies ranging from cardiovascular studies to wound healing and toxicology, as well as agricultural-based research, immunology, and disease studies¹. Blood collected in research can be used to study diseases, pharmacology, genetics, immune system research, and biomarker discovery. Cell culture is one of the most basic procedures done in research and allows researchers to study cell behavior and function, as well as disease models and vaccine development². Cultivation of disease-causing pathogens, such as African Swine Fever Virus, is frequently done in a predetermined culture medium under strict aseptic laboratory conditions. Disease propagation can occur through invasion, spread, or attachment, which require healthy, stable cells³. Pathogens studied may have a specific cell-tropism in which they can be propagated, and having a large volume of blood yields larger amounts of cells to use.

To achieve a high yield of cells from an individual animal, for cellular differentiation, a large volume of blood must be gently collected to maintain cell integrity prior to cell separation. In swine, the total blood volume can be calculated at 65 mL/kg^4,5. In our experience using large volume collection, about half of the total blood volume from an animal can be recovered. Therefore, in large adult swine, weighing up to 600 pounds (273 kg), a yield of 6-8 L is possible. In swine, large volumes of blood can be collected percutaneously from the cranial vena cava, external jugular vein, saphenous vein via cannulation, or the heart^4,5,6. However, intracardiac blood collection with a large-bore cannula allows the largest volume of blood to be efficiently collected with minimal risk for cell lysis. As a terminal procedure, this must be performed under a surgical plane of anesthesia⁵. This article describes a method of using a vacuum pump to collect multiple liters of blood from swine using a percutaneous approach to the heart with a large-bore IV catheter stylet connected to a vacuum pump^7,8.

Protocol

This study was approved by the Midwest Veterinary Services Inc. Animal Care and Use Committee. As the IACUC-approved, primary method of euthanasia during this procedure is exsanguination, it is expected that some pigs will reach cardiac arrest due to blood loss. When this occurs, a secondary euthanasia method, such as bilateral thoracotomy, is performed. After the required blood volume is collected, if there is a palpable heartbeat, a syringe with euthanasia solution is attached to the stylet and administered intracardiac as the primary euthanasia method, followed by a secondary physical method. Swine are maintained in accordance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching, the Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act, as applicable.

1. Preparation of materials and animals (Table of Materials and Figure 1A-C)

1. Assemble the tubing to stainless steel straws and attach the hose end to the bubble tubing.
2. Attach the tubing to the roller bottle using a rubber stopper with two uneven metal straws and a glass bottle.
   NOTE: The glass inhibits the collapse of the container created by the vacuum pump, but any material that does not collapse under pressure can be used.
3. Aseptically instill 10 mL of sodium heparin (1,000 USP/mL) through the suction tubing and the catheter stylet to prevent clotting. Aseptically add 50 mL of sodium heparin (1,000 USP/mL) into each roller bottle to achieve an end concentration of 50 IU/mL in each liter of blood and prevent clotting of the blood during collection. Ensure that the vacuum pump is set to no more than 7 pounds of draw to prevent damage to the cells.

2. Anesthesia and animal positioning

1. Weigh the animal no more than 24 h prior to the procedure to calculate the required dose of the anesthesia combination medications.
2. Inject Telazol (3.0-5.0 mg/kg), ketamine (8-12 mg/kg), and xylazine (4.0-6.0 mg/kg) intramuscularly in the neck, or other large muscle belly (e.g. epaxial muscles), using a 30 inch extension set and 18 G x 1.5" needle to induce deep anesthesia.
   NOTE: In large swine, the maximum recommended IM injection volume is 10 mL, so changing locations after every 10 mL of administration may be necessary⁹. While we chose this cocktail, this procedure can be performed under any IACUC-approved, anesthesia protocol that produces a surgical plane of anesthesia.
3. Once the animal is confirmed to be in a surgical plane of anesthesia (~5-8 min), showing no palpebral reflex, loose jaw tone, and no response to pain stimuli, place the pig on a lift table, floor, or other flat surface, in right lateral recumbency.
4. Clip the hair on the thorax located over the heart, if needed, near the fourth and fifth intercostal space, just caudal to the point of the elbow.
5. If available, use an ultrasound (Figure 2A) to identify the left ventricle. If unavailable, the left ventricle is located nearest the fourth or fifth intercostal space medial to the point of maximal impulse⁸.
6. Aseptically scrub the area with 4 inch x 4 inch gauze sponges soaked in 2% chlorhexidine scrub and 4 inch x 4 inch gauze sponges soaked in 70% isopropyl alcohol. Prep the skin by scrubbing in a circular manner, starting at the point of injection and rotating outwards, for a minimum of 3x, alternating between the two disinfectants.

3. Intracardiac blood collection

1. Verify the animal has remained in a surgical plane of anesthesia by checking palpebral reflex, jaw tone, and pedal reflex.
   NOTE: Check anesthetic depth, as well as heart and respiratory rates, throughout the procedure to ensure the animal remains at a surgical plane of anesthesia.
2. Remove the stylet from the IV catheter. Place the stylet over the left ventricle and advance it perpendicular to the skin to insert the stylet into the left ventricle (Figure 2B). When blood begins to pulse out of the stylet, attach it to the bubble tubing hose end connector that is attached to the roller bottle and then, turn on the vacuum pump (Figure 1A and Figure 2C).
3. Confirm that the blood flows in a steady stream. As blood is pulled into the roller bottle, gently swirl the bottle to allow mixing of the blood with the anticoagulant to prevent clot formation.
4. Once 1 L of blood is collected, use hemostats to clamp the bubble tubing near the roller bottle. Disconnect the rubber stopper and apply it to the next roller bottle. Remove the hemostat and allow blood to flow freely while gently swirling the roller bottle.
5. Continue this process until the blood required has been collected. Once completed, clamp the tubing to prevent excess blood spillage and turn off the pump.
6. Remove the tubing connector from the catheter stylet and attach the sodium pentobarbital syringe to the stylet. Draw back on the syringe to ensure a flash of blood is present then inject the euthanasia solution. Confirm death by the absence of heartbeat, respiration, muscle tone, and reflexes. Once death is confirmed, remove the needle from the heart and place in a sharps container.
7. Dispose bubble tubing in a biological waste container. Remove gross blood from the remaining stainless-steel items (e.g., stainless-steel straws, hose-end connectors) using a tube brush and then autoclave for future use.
8. Place the cleaned rubber stoppers, straws, hose end connectors, and hemostats into a new autoclave pouch and sterilize prior to the next use.

Results

Prior to blood collection, sows were prescreened for Mycoplasma hyopneumoniae, porcine adenovirus, porcine parvovirus, swine coronaviruses (transmissible gastroenteritis, porcine epidemic diarrhea), porcine hemoagglutinating encephalomyelitis, porcine reproductive and respiratory syndrome, influenza group A, circovirus 1 & 2, and Seneca virus A. We have repeated this procedure multiple times in female Yorkshire cross sows weighing 300-600 lbs. Blood collection yielded between 3 L from the smaller swine to 8 ...

Discussion

The procedure here describes a catheter-stylet-to-vacuum-pump assembly for collecting a one-time large volume of blood from the heart of sows. Because of the potential for pain and distress, this procedure must only be performed in animals that are in a surgical plane of anesthesia. Immobility from the anesthesia allows this procedure to be learned by beginners and mastered quickly. Multiple veins can be used to collect blood from animals. The most common and accessible routes are the jugular veins, cranial vena cava, an...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1/8 HP Lab Model Diaphragm Pump – 115 vac, 60 max psi	Base Supply Center	80058845
10 G x 6 in catheter-over-needle	Covetrus North American Veterinary Supplies	024616
50 mL syringe, luer lock	Fisher Scientific	13-689-8
Aquasonic 100 ultrasound gel blue	Midwest Veterinary Supply	001.02109.2
Argyl bubble tubing, Nonconductive (¼")	Fisher Scientific	NC2282605 (Vendor catalog # 8888280412)
Chlorhexidine Q Scrub 2%	Midwest Veterinary Supply	562.30804.3
Corning Disposable Roller Bottles.	Fisher Scientific	06-413
Corning PYREX narrow mouth heavy-duty glass Erlenmeyer flask or similar creating suction	Fisher Scientific	10-040F
Eisco nylon test tube brush	Fisher Scientific	S13962
Fisherbrand two-hole rubber stoppers	Fisher Scientific	Dependent on size
Ketamine (10 mg/mL)	Midwest Veterinary Supply	275.22100.3
MLL to 6 mm hose end (stainless steel)	Cadence Science	6547IND
MLL to 8mm hose end (stainless steel)	Cadence Science	6549IND
MVet gauze sponge non-woven 4 x 4 4 ply	Midwest Veterinary Supply	000.30156.2
MVet isopropyl alcohol 70%	Midwest Veterinary Supply	000.50054.3
MVet Kelly hemostatic forceps straight 5.5”	Midwest Veterinary Supply	000.36800.2
Sodium Heparin (1000 U/mL)	Midwest Veterinary Supply	191.46701.3
Sodium Pentobarbital (390 mg/mL)	Midwest Veterinary Supply	749.20000.3
Stainless steel straws	Hobby Lobby	1893635
Telazol (100 mg/mL)	Midwest Veterinary Supply	275.40000.3
Xylazine (100 mg/mL)	Midwest Veterinary Supply	310.01150.3