A method for collecting high numbers of blood samples in standard vacuum tubes from non-heparinized pigs

Abstract

Large animal models allow for collection of substantial amounts of biological material. However, the collection of larger volumes (>100 ml) of blood from pigs can be a challenge: (i) the peripheral veins are not suitable for collection of high numbers of standard blood tubes as the veins tend to collapse; and (ii) the alternative option of cannulating deeper veins mandates surgical exposure of the vessels and often the need for heparinization, which is undesirable for some blood analysis. During an immunization trial in 40 pigs, we assessed the femoral and saphenous arteries as practical vessels for collecting 250 ml of blood from each pig in standard collection tubes without heparinization. Blood collected from the saphenous artery by a standard butterfly needle proved particularly useful and 250 ml blood could be collected successfully in 24 of 25 pigs by this approach.

The pig is a favoured large animal model for human biomedical research because it recapitulates important aspects of human disease and its size provides abundant access to biological material.^1,2 In particular, the substantial blood volume of pigs facilitates repeated blood sampling or collection of large samples that is important for analysis and monitoring of basic blood parameters. Many analyses can be performed in small volume samples, but larger amounts of blood may be desired for particular studies, for example, functional neutralization assays, in vitro flow-chamber model systems or ex vivo kidney perfusion studies.^3,4 Collection of larger quantities of blood, however, can be troublesome as it often requires access to deeper veins, which mandates surgical exposure of the vessel, requiring general anaesthesia and heparinization to prevent coagulation. The latter may not always be desirable as heparin content in blood samples may affect analysis of key functions of the blood, including coagulation propensity, complement ⁵ and contact activation system. ⁶ The femoral and saphenous arteries are described as accessible vessels for arterial cannulation, but detailed description and implication of this procedure in non-heparinized animals is limited.^7,8

The aim of this study was to assess the femoral and saphenous arteries as practical routes to collect 25 standard blood tubes (roughly 250 ml blood per animal) without the need for heparinization. Blood samples were collected from 40 pigs in a project that required pig serum for further laboratory experiments. The project was approved by the Danish Animal Experiment Inspectorate, licence number: 2021-15-0201-00821. No animals were excluded from the study.

Female Landrace × Yorkshire pigs were sourced commercially (Kokkenborg ApS, Denmark) and acclimatized for at least one week before the experiments began. They were housed in communal enclosures (3 m² per animal) with sawdust bedding and daily enrichment in the form of various toys. Pigs were attended at least twice daily and given a standard feed (Prime Star B, DLG) and free access to water. The average bodyweight was 55 kg (SD: 4.3) at the time of the procedure.

For all blood sampling procedures, animals were deeply sedated using a mixture of one vial of dry matter zolazepam/tiletamine (Zoletil 50 Vet., Virbac Danmark, Kolding, Denmark) dissolved in 6.45 ml xylazin (Sedaxylan Vet., 20 mg∙ml⁻¹, Dechra Veterinary Products, Uldum, Denmark), 1.25 ml ketamine (Ketaminol Vet., 100 mg∙ml⁻¹, MSD Animal Health, Copenhagen, Denmark), 2 ml butorphanol (Butomidor Vet., 10 mg∙ml⁻¹, Salfarm Danmark, Kolding, Denmark) and 2 ml methadone (Insistor Vet., 10 mg∙ml⁻¹, Salfarm Danmark, Kolding, Denmark). Each pig was administered 1.3 ml /10 kg body weight of the mixture intramuscularly. The procedure was initiated when complete muscle relaxation and absence of reflexes was reached. To secure adequate sedation depth, muscular reflexes were monitored frequently. Cardiovascular and respiratory function was monitored with pulse oximetry (heart rate and blood oxygen saturation) and observation of respiration pattern, respectively, and oxygen administered via a face mask to prevent hypoxia. Since the procedure was non-recovery, local anaesthetics were not applied, and no indication of pain was observed during the procedure. All animals were euthanized at the end of the procedure with 5 ml i.v. pentobarbital (200 mg∙ml⁻¹).

Procedure 1: femoral artery catheterization

In 15 pigs, blood was collected through a catheter placed in the femoral artery. To do so, the pig was placed in supine position on the operating bed and the hind legs relaxed. The femoral artery was palpated below the inguinal ligament; however, arterial pulse was not always palpable, in which case the location was based on the anatomy of the inguinal ligament at approximately one-third of its length from the medial attachment. When the anatomical location was identified, surgical cutdown was performed by an oblique skin incision, followed by arterial exposure of the deep femoral artery which lies just lateral to the femoral vein (Figure 1). A 16G central venous catheter (CV04301, ARROW) was inserted over a 0.81 mm guidewire using the Seldinger Technique while the cavity was held opened using a self-retaining retractor. It was important to insert the catheter completely to avoid dislodgement. From the lure-lock tip of the catheter, a 20 ml syringe was used to aspirate blood, which was immediately transferred to clot activator tubes (10 ml, BD Vacutainer) by an assistant. Blood aspiration was performed uninterrupted using three syringes, to avoid clogging of the catheter by coagulation.

Figure 1.

The femoral artery (arrow) was exposed caudal of the inguinal ligament and just lateral and superior to the femoral vein (a). The cavity is held open using a self-retaining retractor and a catheter is inserted in the artery (b).

Outcome

The procedure was successful in 86% of pigs (13 of 15) as the catheter was dislodged in two animals. In one of these, however, the catheter could be re-inserted and the procedure finished. Although blood was continuingly aspirated, coagulation could not be completely prevented inside the syringes. The procedure was technically challenging as the deep location of the artery mandated vessel-puncture in a near-perpendicular angle.

Procedure 2: arterial puncture of the saphenous artery

In 25 pigs, we explored an alternative approach, in which the saphenous artery of the hind leg was exposed to facilitate blood collection using a standard 21G butterfly needle (Safety-Lok, BD Vacutainer). The pigs were placed on their left side and the artery palpated on the medial side of the left hind leg. Approximately 5 cm from the groin, at a location where arterial pulse was clearly palpable, a 30 mm skin incision was made and the artery exposed by subcutaneous dissection (Figure 2). It is important not to expose the artery further, as the surrounding connective tissue and fascia fixates the artery thereby making it easier to place the needle correctly. When the artery was exposed, the butterfly needle was inserted in the direction of the black arrow (Figure 2(b)). One person held the needle in place, while another person collected the blood in tubes. A schematic drawing of the anatomic references is shown in Supplementary material Figure 1 online.

Figure 2.

The saphenous artery was exposed by subcutaneous dissection (a). Arterial puncture in the direction of the black arrow using a 21G butterfly needle facilitated collection of 25 standard 10 ml blood tubes.

Outcome

The approach was successful in 96% of pigs (24 of 25). The saphenous artery was consistently palpable. In one pig, the artery collapsed and the procedure was attempted on the contralateral side, where the procedure was also unsuccessful.

Discussion

In summary, the saphenous artery is an easy and reliable access route to collect high numbers of blood samples in standard blood tubes. The procedure requires only minimal materials (needle and tubes) and can be performed without significant training or surgical experience of the operator and without heparinization of the animal. Catheterization of the femoral artery without heparinization proved to be an inferior, more costly and more technically demanding procedure.

Blood collected by both approaches could be processed by standard means (i.e. effective coagulation in the clot-activating tube and centrifugation) without visual haemolysis or fibrin formation, indicating that the quality of the collected material was not significantly influenced by the procedure or tranquilization cocktail compared with regular blood samples. We collected 250 ml blood in a single draw, corresponding to roughly 7.5% of the total blood volume. We did not investigate whether higher volumes could be collected or how often; however, it is not unlikely, that the incision could be sutured to allow for repetitive sampling after a relevant recovery period. Hence, this collection method might be considered for procedures with recovery, although only with further precautions such as the use of aseptic techniques. An alternative, and possibly less invasive, approach is percutaneous catheterization of the saphenous artery, which has been proposed by Namba et al. ⁸ Namba and colleagues, however, conclude that the thick epidermis made the procedure technically challenging and suggest that a small incision and subcutaneous dissection is performed prior to needle puncture; hence, a surgical approach to expose the vessel seems to be the most reliable approach for accessing the saphenous artery. Here we used relatively large animals with a mean weight 55 kg, and it is not unlikely that our approach for saphenous artery access may be less successful in smaller individuals. From a perspective of the 3Rs, it is likely that the saphenous artery may be accessed, as proposed in this study, under lighter sedation supplemented with local analgesia to refine the procedure. We chose a deep sedation to facilitate direct comparison with procedure 1 (i.e. femoral catheterization).

Conclusion

Arterial puncture of the saphenous artery is a convenient approach for collecting large blood samples in non-heparinized pigs.

Supplemental Material

sj-pdf-1-lan-10.1177_00236772221133431 - Supplemental material for A method for collecting high numbers of blood samples in standard vacuum tubes from non-heparinized pigs

Supplemental material, sj-pdf-1-lan-10.1177_00236772221133431 for A method for collecting high numbers of blood samples in standard vacuum tubes from non-heparinized pigs by Kristian Stærk, Louise Langhorn, Yaseelan Palarasah and Thomas E Andersen in Laboratory Animals