Validity of a novel ex vivo porcine liver perfusion model for studying haemostatic products

Abstract

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Introduction

We developed a novel normothermic ex vivo porcine liver perfusion model with whole blood in order to have alternatives for animal experiments in the research and development of new local haemostatic agents. This study aims to assess the construct and content validity of this model.

Methods

In this study we performed two ex vivo experiments using nine livers and one in vivo experiment using six female Norsvin Topigs pigs: (1) ex vivo liver perfusion for establishing physiological blood parameters of the perfused liver and controlled heparinization, (2) ex vivo liver perfusion with a surgical injury and (3) a surgical liver injury in anaesthetized pigs with and without heparin.

Results

Ex vivo coagulation parameters were comparable to in vivo with heparin. Blood gas values and metabolic parameters were comparable between ex vivo and in vivo with heparin, but significantly different compared with in vivo baseline, with the exception of (partial pressure of oxygen (PO₂). Activated clotting time (ACT) values significantly differed depending on the heparin doses. The coagulation parameters fibrinogen, activated partial thromboplastin time, prothrombin time and ACT were rather constant during the 4 h ex vivo perfusion. Haemostatic efficacy of commercially available products was comparable between in vivo with heparin and the ex vivo liver perfusion experiment.

Conclusion

This novel ex vivo liver perfusion model demonstrates good construct and content validity for at least 4 h of perfusion. The model is an easily accessible, table-top, tunable and effective alternative for the in vivo testing of (new) haemostatic products on their haemostatic properties.

Validité d'un nouveau modèle de perfusion hépatique porcin ex-vivo pour l'étude des produits hémostatiques Résumé

Keywords

Alternatives surgical bleeding pigs liver ex vivo haemostasis sealant sealing patch TachoSil Veriset Hemopatch

Introduction

Bleeding is an inevitable part of an operation, but when moderate or severe it can lead to longer operative time, a higher risk of reintervention, more postoperative complications, a prolonged hospital stay, more readmissions and increased healthcare costs.^1–3 Most bleedings are minor and can be stopped by packing with gauze, electrocautery or suturing. In some instances, depending on the bleeding location or tissue, bleeding needs to be dealt with with haemostatic agents.^4,5 Numerous topical haemostatic agents have been developed to achieve haemostasis during surgery, varying from patches to glues and sprays.^6,7 Although generally effective, most commercially available haemostatic agents have the important limitations of having little effect with coagulation disorders and use of anti-coagulant drugs, not being able to address severe bleeding, application difficulty in minimally invasive surgery, need of human or animal derived components, and high manufacturing costs.⁸

Medical device regulation demands evaluating a new haemostatic product for efficacy and safety in animals before use in patients. Dependent on the application site, different animal species and experimental models are used. Rats,^9–11 rabbits^12,13 and pigs^14,15 are dominant in common experimental models that include liver resection and injury,^12–15 arterial bleeds^9,13 and partial nephrectomy.¹⁰ In the last five years, more than 300 peer reviewed papers on haemostatic agent studies in animals were published annually. A conservative estimate of 40 animals per study means that over 10,000 animals are sacrificed every year for the preclinical testing of haemostatic agents. Most likely the actual number of animals is a multiple of 10,000, because animals used by commercial animal facilities for haemostat prototype selection, early efficacy and biocompatibility are often not included in scientific publications.

Within the context of the three Rs (Reduction Replacement Refinement)¹⁶ in animal research, there is an increasing need for alternatives to replace or to reduce the number of animals in experiments. For development and testing of haemostatic products, alternatives might particularly be an option in the phases of prototype selection, handling characteristics and early safety and efficacy. Based on our experience with normothermic ex vivo machine perfusion for testing and priming organs for transplantation,^17,18 we started a programme of ex vivo organ perfusion for early research and development of surgical biomaterials. A key part of this programme is the mimicking of common physiology (blood pressure, blood flow, temperature and coagulation parameters) as well as pathophysiological conditions for better translational value, which often is not possible in animals. In the context of haemostat research, the model should allow for changes of the perfused liver that resemble clinical conditions relevant for haemostat application, such as coagulation disorders induced by anti-coagulant drugs or massive bleeding. Last, such a model must be easy to deploy, provide easy access for studying different formulations of haemostatic products including patches, powders, sealants and sprays, and must be cost-effective.

In this study, we validate our ex vivo liver perfusion model of surgical liver injury for its construct of usability and efficacy of haemostatic products and for its content of similarity with using these products in vivo. A surgical liver injury model is relevant because liver surgery is an increasingly frequent procedure, with use of haemostatic products in about 50%.¹⁹

Methods

Design

In this study we performed two ex vivo and one in vivo experiment: (1) ex vivo liver perfusion for establishing physiological blood parameters of the perfused liver and controlled heparinization, (2) ex vivo liver perfusion with a surgical injury and (3) a surgical liver injury in anaesthetized pigs with and without heparin. Regular blood samples were taken from the perfusion system for chemistry, gas and haematology analyses in all experiments. Blood parameters were compared between the ex vivo perfusion and the in vivo situation. Usability and efficacy of three commercially available haemostatic patches applied on the surgical liver defects were compared between the injured ex vivo and in vivo livers.

Ex vivo perfusion experiments

A standard extra corporeal organ procurement system (ECOPS; Organ Assist, Groningen, the Netherlands) with a disposable set for extracorporeal organ perfusion was used. The set was adapted by placing an extra reservoir with an overflow safeguard between the oxygenator and the liver in order to keep the blood pressure in the supplying tube at 5–10 mmHg, as shown in Figure 1.

Figure 1.

Schematic representation of the adjusted ECOPS setup.

Livers (N = 9) and blood were obtained from a slaughterhouse. After being stunned with an electric current applied with electrodes on the body as a routine procedure for meat processing, the carotid artery was cut open to exsanguinate the pigs (Sus scrofa domesticus, the Netherlands). Blood was collected directly from the carotid artery wound and stored in a plastic container, which was prepared with 1000 IE heparin per litre of blood (Leo Pharma, Ballerup, Denmark). Ten litres of blood was collected from multiple pigs. To harvest the liver, the abdomen was opened via a midline incision. The liver was excised with attached blood vessels and part of the diaphragm, placed immediately in a plastic bag on melting ice and transported to the surgical laboratory. Blood was stored at ambient temperature during transportation.

The ECOPS was filled and primed with the heparinized blood, which could circulate through a bypass. The liver was removed from the plastic bag and placed on a plastic sheet, floating in a water bath, and connected to the ECOPS by cannulating the portal vein and vena cava. Next, a mixture of 95% O₂ and 5% CO₂ was used to oxygenate the blood (flow = 2.5 l/min). The temperature of the blood and the water bath were set at body temperature (37°C). The temperature of the blood was maintained by the ECOPS on 37°C using the heat exchanger in the oxygenator, measuring it with a temperature probe. The water bath was kept at 37°C using a thermostat. The liver was packed with gauzes soaked in saline to prevent desiccation.

After priming the system, blood was allowed to circulate through the liver at a minimal rate of 500 ml/min and a pressure of 5–10 mmHg until return to normal colour and a temperature of the blood returning from the liver (proxy core liver temperature) above 36°C, before starting the experiment.

After pH normalization and control for colour and temperature of the perfused liver, standard lesions were made for studying haemostasis with one of three haemostatic patches: Veriset™ (Covidien Inc., Mansfield, MA, USA), TachoSil® (Takeda Austria GmbH, Linz, Austria) and Hemopatch (Baxter AG, Vienna, Austria). These patches were chosen due to their common application in liver surgery.

The lesions were punch holes made with a skin biopsy punch (Ø 8 mm; KAI Medical, Solingen, Germany) with a 3 mm thick rubber ring to standardize the depth of the biopsy. The bleeding rate in g/min was quantified by placing a dry pre-weighed gauze below the lesion, waiting for 30 s, and removing and weighing the gauze. Immediately, one of three patches was applied using a gauze and digital pressure according to the instructions of use (1 min for Veriset™ and Hemopatch, and 3 min for TachoSil®). After removing the gauze, the products were observed for 3 min. Outcome was defined as a success if bleeding was stopped within this 3-min timeframe. Nine lesions were made on each liver to measure time to haemostasis. Patches were applied in alternating order.

In vivo experiment

Six healthy, MRSA free female pigs (40–60 kg, Sus scrofa domesticus, Norsvin Topigs, age: 2–3 months) were used to collect blood samples and to study efficacy of haemostatic patches. The animals were obtained from a local farm and were housed per pair in a pen with water and standard pig chow (Professional Pig Feed, Havens, Maashees, Netherlands (NL)) ad libitum with an acclimatization period of at least five days. A standard 12-h light/dark cycle was maintained with room temperature of 20°C. The pens were embedded with straw and enriched with toys. Pigs were previously used for testing haemostatic agents and prior to sacrifice, when the animals were anaesthetized and all measurements for the initial study were executed, we were allowed to perform the described experiment. The animals were included only when mean arterial pressure was above 60 mmHg.

The animals received premedication consisting of meloxicam (24 h prior to surgery; Boehringer Ingelheim, Ingelheim am Rhein, Germany) and ketamine (10 mg/kg; Alfasan, Woerden, Breda, NL), midazolam (1 mg/kg; Roche, Basel, Switzerland) and atropine (50 µg/kg; Centrafarm, Breda, NL) 15 min before surgery. Antibiotics (amoxicillin, 20 mg/kg; Centrafarm, Breda, NL) were given just before anaesthesia. Pigs were anaesthetized using propofol (2.5 mg/kg; Fresenius, Bad Homberg vor der Höhe, Germany) according to the standard protocol of the animal research facility (see Supplementary material Table S1 online). During surgery the anaesthesia was maintained by isoflurane (0.5%; Pharmachemie BV, Haarlem, NL) in combination with midazolam, sufentanil (Forte, Janssen, Beerse, Belgium), meloxicam and atropin. Rocuronium was given when muscular contraction appeared. Blood samples were taken from an arterial pressure line that was placed at the start of the surgery in the left femoral artery. After disinfection of the abdomen, sterile drapes were placed. All pigs underwent midline laparotomy using diathermy and after the liver was exposed, the lobe was mobilized and, if necessary, held in position using a large pre-soaked abdominal gauze. Mean arterial pressure was maintained above 60 mmHg. Lesions were made, 3 mm deep and comparable to the ex vivo procedure for testing the haemostatic products. The bleeding rate measurement and application of the products was similar as described for the ex vivo perfusion experiment. The surgeon was blinded for the product until after creating the bleeding and products were allocated in a random way divided between the pigs, using the Microsoft Excel Aselect() function. Pigs were euthanized with an overdose of pentobarbital (AUV, Cuijk, NL) at the end of the surgery in this non-survival experiment. The study was approved by the Dutch Animal Ethics Committee (CCD: AVD-10300 2015 348) and performed in the Central Animal Laboratory of the Radboud University. The protocol was reviewed by the Animal Welfare Body of the Radboud University.

Blood sampling and analyses

In the ex vivo experiment, blood samples were taken immediately at the start and at intervals of 30 min after temperature and pH normalization, until termination of the experiment. When the pH was lower than 7.35 at first blood gas analysis, 1 M bicarbonate was titrated to the system until pH reached a value between 7.35 and 7.45. Blood gas, glucose, pH, sodium (Na), potassium, ionized calcium (iCa), base excess in the extracellular fluid compartment (BEecf), haematocrit (Hct), haemoglobin (Hb), bicarbonate (HCO₃) and activated clotting time (ACT) were analysed with the I-STAT (Abbott Point of Care Inc., Princeton, NJ, USA) portable clinical analyser with CG8+® and ACT-K cartridges (Abbott Point of Care Inc., Princeton, NJ, USA). An EDTA blood collection tube was used to sample blood for measuring activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen, erythrocytes, thrombocytes, leucocytes, mean corpuscular haemoglobin concentration, mean corpuscular volume, mean corpuscular haemoglobin, red cell distribution width (RDW), Hct and Hb were analysed in the routine clinical laboratory. At the first and last sample time an additional blood sample tube (sodium citrate) was used for rotational thromboelastometry (ROTEM), clotting time (CT), clot formation time (CFT) and maximum clot firmness (MCF).

In the in vivo experiment, blood was sampled for baseline levels (without heparin) of coagulation parameters, ROTEM, haematology, blood gas values and metabolic parameters. Before the first lesion was made, heparin was administered (300 U/kg) with the goal of reaching an ACT of 2–3 times baseline. When the ACT level was below 2 × baseline, additional heparin was titrated until target was reached. Blood was collected before the first and after the last haemostatic product was tested.

For the comparison between the ex vivo, the in vivo baseline and the in vivo with heparin, multiple blood samples were taken. During the ex vivo experiment, for controlling the constancy of blood parameters, blood samples were taken at the beginning of normo-temperature perfusion, and subsequently every 30 min. During the in vivo experiment, blood samples were taken at the beginning of the surgery for the baseline (without heparin) values. For the in vivo with heparin part, samples were taken after administering heparin, before applying the first product and at the end of the experiment.

Statistical analysis

We did not perform an a priori sample size calculation for various reasons, including reuse of pigs, only including pigs with a mean arterial blood pressure of 60 mmHg and the variability in available space on the liver to create and treat a bleeding injury. However, from previous experiments we know that numbers of lesions between eight and 12 have sufficient power.

Statistical analyses were performed using SPSS v.25 (IBM, Armonk, NY, USA) software. For the overview of all measured parameters, means and standard deviations were calculated (Table 1). Comparison of bleeding rate, coagulation parameters, ROTEM, haematology, blood gas values and metabolic parameters were done using an analysis of variance with a Tukey post-test, with p-values corrected for multiple comparisons. Success rate of the different haemostatic patches was compared between ex vivo and in vivo with heparin using a chi square–Fisher’s exact test, with a p-value <0.05 indicating statistical significance. Comparisons over time for the coagulation parameters (aPTT, PT, fibrinogen and ACT) were performed using Kendall’s tau trend test.

Table 1.

Overview of all measured parameters in the ex vivo liver perfusion model and the in vivo baseline and with heparin conditions in the anaesthetized pig.

		Ex vivo perfusion			In vivo baseline			In vivo with heparin
		n	Mean	SD	n	Mean	SD	n	Mean	SD
Coagulation
ACT	(s)	38	348	15	10	89	4	16	221	13
aPTT	(s)	35	47	5.7	1	20	–	8	60	7.8
Fibrinogen	(mg/l)	37	2070	39	23	1598	82	8	1613	353
PT	(s)	37	16	0.2	23	15	0.1	8	16	0.3
ROTEM
EXTEM-CT	(s)	10	60	6.6	5	48	3.0	3	53	4.1
EXTEM-CFT	(s)	10	96	30.0	5	43	1.6	3	42	1.0
EXTEM-MCF	(mm)	10	56	3.8	5	69	1.9	3	75	1.5
INTEM-CT	(s)	10	4696	689	5	130	6.1	3	4934	599
INTEM-CFT	(s)	10	637	–	5	50	5.1	–	–	–
INTEM-MCF	(mm)	10	20	15	5	64	2.9	–	–	–
HEPTEM-CT	(s)	10	410	70.1	5	127	6.7	2	201	6.5
HEPTEM-CFT	(s)	10	355	164	5	60	6.4	2	57	9.5
HEPTEM-MCF	(mm)	10	49	4.1	5	61	3.1		68	3.5
Haematology
Erythrocytes	(10¹²/l)	36	9.2	0.2	23	5.3	0.1	8	5.5	0.3
Thrombocytesees	(10⁹/l)	28	95	7.4	23	280	17.3	8	286	15.9
Leucocytes	(10⁹/l)	36	6.5	0.4	23	14.2	0.9	8	17.6	1.3
MCHC	(mmol/l)	36	17.4	0.2	23	18.6	0.1	8	18.4	0.1
MCV	(fl)	36	64	0.4	23	60	0.5	8	61	0.9
MCH	(amol)	36	1.11	0.01	23	1.11	0.01	8	1.12	0.01
RDW	(%)	28	20.6	0.2	23	15.9	0.2	8	15.9	0.4
Hct	(l/l)	36	0.59	0.01	23	0.32	0.01	8	0.34	0.01
Hb	(mmol/l)	36	10.3	0.2	23	5.9	0.1	8	6.2	0.3
Blood gas and metabolic values parameters
pCO₂	(kPa)	40	5.9	0.3	22	5.4	0.1	8	5.5	0.2
pO₂	(kPa)	40	75.1	0.9	22	33.6	1.5	8	29.8	3.1
BEecf	(mmol/l)	40	0.7	1.9	22	8	0.5	8	6	0.7
HCO₃	(mmol/l)	40	26.0	1.6	22	30.8	0.4	8	28.8	0.6
TCO₂	(mmol/l)	40	27	1.6	22	32	0.4	8	30	0.6
sO₂	(%)	40	100	–	22	100	0.045	8	100	0.1
pH		40	7.37	0.03	22	7.49	0.01	8	7.46	0.02
Na	(mmol/l)	40	139	1.2	22	140	0.3	8	140	0.4
K	(mmol/l)	40	x	–	22	4	0.1	8	5	0.1
iCa	(mmol/l)	40	0.69	0.03	22	1.35	0.02	8	1.35	0.02
Glucose	(mmol/l)	27	17.04	2.1	22	4.2	0.3	8	4.7	0.6

SD: standard deviation; ACT: activated clotting time; aPTT: activated partial thromboplastin time; PT: prothrombin time; ROTEM: rotational thromboelastometry; CT: clotting time; CFT: clot formation time; MCF: maximum clot firmness; MCHC: mean corpuscular haemoglobin concentration; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; RDW: red cell distribution width; Hct: haematocrit; Hb: haemoglobin; pCO₂: partial pressure of carbondioxide; pO₂: partial pressure of oxygen; BEecf: base excess in the extracellular fluid compartment; TCO₂: total carbondioxide; sO₂: oxygen saturation; iCa: ionized calcium; x: out of range .

Results

Harvesting of the liver in the slaughterhouse was completed on average 30 min after cardiac arrest with a maximum of 40 min. Transportation time to the surgical laboratory was on average 45 min with a maximum of 1 h. Five ex vivo liver perfusion procedures were performed for construct validation of the model. Three perfusions could be continued for 4 h and two for 3.5 h. In all livers, colour and temperature had returned to about normal within 30 min after start of the perfusion (see Supplementary Figure S1). Four livers were used in the injury experiment and no complications during the procedure were observed. The surgery in the in vivo experiment went uneventfully in all pigs, took between 1.5 h and 2 h, and all measurements were done with a mean arterial pressure above 60 mmHg.

Comparison of blood parameters between ex vivo and in vivo baseline and with heparin

Table 1 provides an overview of the blood values.

Coagulation parameters

The mean ex vivo aPTT was comparable to in vivo with heparin (47 s and 60 s, p = 0.403) and both showed higher values compared with in vivo baseline (20 s) values. Ex vivo ACT (348 s) was higher (p = 0.000) compared with in vivo with heparin (221 s), and both were significantly higher compared with in vivo baseline (89 s, p = 0.000; Figure 2(a)). The PTs (Figure 2(b)) did not differ between ex vivo and in vivo with heparin (16 s and 16 s, p = 0.141). Ex vivo PT was higher compared with in vivo baseline (15 s, p = 0.000). Ex vivo fibrinogen amount (2070 mg/l; Figure 2(c)) was higher compared with both in vivo conditions (1598 and 1613, p = 0.000 and p = 0.025, respectively).

Figure 2.

Comparison of coagulation parameters ex vivo, in vivo baseline and with heparin.

ROTEM

The results of the ROTEM test showed no coagulation and clot formation time in the ex vivo perfusion and in vivo with heparin condition for the INTEM test. After adding heparinase (HEPTEM test) mean ex vivo CT was higher compared with in vivo baseline (410 s and 127 s, p = 0.029) but not with in vivo with heparin (201 s, p = 0.311). The EXTEM showed comparable CT and CFT for all groups. Ex vivo MCF was lower compared with in vivo with heparin (49 and 68 mm, p = 0.025).

Haematology

The mean number of thrombocytes (95*10⁹/l) and leucocytes (6.5*10⁹/l) ex vivo was lower (both p = 0.000) compared with in vivo baseline (280*10⁹/l and 286*10⁹/l for thrombocytes and 14.2*10⁹/l and 17.6*10⁹/l for leucocytes). In contrast, the number of erythrocytes (9.2*10¹²/l, 5.3*10¹²/l and 5.510¹²/l, p = 0.000) and the RDW (20.6, 15.9 and 15.9, p = 0.000) was increased (Figure 3).

Figure 3.

Comparison of haematology parameters ex vivo, in vivo baseline and with heparin: (a) thrombocyte count; (b) leucocyte count; (c) erythrocyte count and (d) red cell distribution width (RDW).

Blood gas values and metabolic parameters

Means of ex vivo partial pressure of oxygen (PO₂) (75.1, 33.6 and 29.8 kPa, p = 0.000), Hct (0.59, 0.32 and 0.34 l/l, p = 0.000), Hb (10.3, 5.9 and 6.2 mmol/l, p = 0.000) and glucose (17.04, 4.2 and 4.7 mmol/l, p = 0.000) were higher compared with in vivo baseline and with heparin. iCa (0.69, 1.35 and 1.35 mmol/l, p = 0.000) and BEecf (0.7, 8 and 6 mmol/l, p = 0.018) were significantly lower compared with both in vivo conditions. Mean pH was lower than in vivo baseline (7.37 and 7.49 p = 0.007), but not different (7.46, p = 0.257) from in vivo with heparin. Potassium was undetectable in the ex vivo blood samples.

Means of partial pressure of carbondioxide (PCO₂) (5.9, 5.4 and 5.5 kPa, p = 0.413), HCO₃ (26.0, 30.8 and 28.8 mmol/l, p = 0.055), total carbondioxide (TCO₂) (27, 32 and 30%, p = 0.073), oxygen saturation (sO₂) (100, 100 and 100%, p = 0.132) and Na (139, 140 and 140 mmol/l, p = 0.973) showed no significant differences between ex vivo and the in vivo conditions.

Constancy in time

The PT and amount of fibrinogen remained constant during the course of the perfusion (Figure 4). The aPTT showed an initial decrease (from 113 s to 67 s) and little variation thereafter until the end of the experiment. ACT remained about constant with a level three times the baseline for the duration of the experiment.

Figure 4.

Coagulation parameters over time in the ex vivo perfusion experiment: (a) fibrinogen concentration; (b) activated partial prothrombin time (aPTT); (c) prothrombin time (PT) and (d) activated clotting time (ACT).

Comparison of haemostatic agents between ex vivo and in vivo

Injuries and the haemostasis after product application were compared between the ex vivo perfused liver and the in vivo with heparin condition in the anaesthetized pig. The severity of the bleeding was comparable for all lesions; mean (±SD) bleeding rates of 3.7 ml (±2.9)/30 s for ex vivo and 3.7 ml (±1.0)/30 s for in vivo. Veriset™ showed 100% haemostatic efficacy in vivo compared with 62.5% ex vivo (p = 0.066; Figure 5). Efficacy of TachoSil® was low without differences between models (9% ex vivo and 12.5% in vivo, p = 1.000). Hemopatch performed moderately with percentages of 55.6% ex vivo and 44% in vivo, p = 1.000.

Figure 5.

Haemostatic success of three different patches.

Discussion

This novel ex vivo liver perfusion model for research purposes is a valid alternative for the in vivo testing of products regarding haemostatic efficacy. We showed that the most relevant blood values are comparable to in vivo measurements and remain constant during the perfusion over the course of at least 4 h. Commercially available local haemostatic products showed comparable efficacy in both the high and the low ranges. However, Veriset™ underperformed in the ex vivo perfused liver model in comparison with performance in the in vivo model

The most important parameters, pH, aPTT and PT, did not differ between ex vivo and in vivo and remained constant during the perfusion time. However, the HEPTEM test showed an elevated CT for the ex vivo perfusion model compared with the in vivo baseline condition, as well as an increased CFT, due to the administration of heparin in the ex vivo perfusion model. This is supported by the higher ex vivo ACT level. Although the MCF in the EXTEM test was lower in the ex vivo perfusion, the value was within the clinically relevant range, showing clot formation. Furthermore, no differences were found between CT and CFT in the EXTEM test.

The increased amount of erythrocytes is possibly due to an inadequate distribution in the main reservoir in the ex vivo setup, causing the erythrocytes to sink to the bottom of the reservoir and into the loop. This can also explain the increased Hct and Hb levels. Continuously mixing the blood in the reservoir may have prevented this imbalance.

Thrombocytes might have been damaged and started to clot due to the centrifugal pump. These clots are probably caught in the filter of the oxygenator, causing the decreased amounts in the ex vivo perfusion model. Similar may have accounted for the low leucocyte concentrations. Despite the decreased amount of thrombocytes and increase of erythrocytes, coagulation was within normal range, as shown by corresponding CT, CFT and MCF values in the EXTEM test.

After an initial drop, aPTT levels were stable for the remainder of the perfusion, as were ACT levels three times above baseline, fibrinogen and PT. These findings indicate that testing of local haemostatic products for up to 4 h using this model should be feasible without risk of bias due to prolonged perfusion time.

Observed differences in other parameters such as PO₂, BEecf and glucose levels were possibly due to methodological requirements of ex vivo liver perfusion. However, these parameters hardly affect the working action of local haemostatic products. pH levels were maintained between the targeted normal range of 7.35 and 7.45, assuring constancy for this relevant, haemostasis affecting parameter. Potassium levels were not measurable in the ex vivo blood samples, due to the amount of heparin and the storage of blood.²⁰

The comparable haemostatic effect of commercially available local haemostatic products in stopping the bleeding of a surgical liver injury indicates construct and content validity of the ex vivo perfusion model. Furthermore, findings suggest potential clinical relevance of this model in challenging conditions of liver surgery such as in patients with coagulation disorders. In vivo and ex vivo using Veriset™ showed the highest haemostatic efficacy. Veriset™ consists of an oxidized regenerated cellulose (ORC) backing, a trilysine layer and a coating with polyethylene glycol (PEG). The PEG will adhere to the tissue while the trilysine layer with blood forms a hydrogel barrier. Because Veriset™ adheres well to tissue, the haemostatic efficacy is potentially higher than that of TachoSil® and Hemopatch. While having the same polymer, difference between Veriset™ and Hemopatch can be explained by the different carrier, ORC with trilysine compared with collagen. In the in vivo experiment the efficacy was 100% for Veriset™ compared with 60% in the ex vivo perfusion model, as determined 3 min after application. However, when assessing success after 5 min, Veriset™ demonstrated 100% success in the ex vivo model. The difference between 3 and 5 min assessment is probably caused by the excess of heparin used ex vivo, in which condition more time is needed for crosslinking and coagulation.

Results show a comparable low and moderate efficacy for TachoSil® and Hemopatch in both ex vivo and in vivo experiments. The haemostatic working mechanism of TachoSil® is based on activation of fibrinogen and thrombin. As heparin prevents coagulation by inactivating thrombin and the conversion of fibrinogen to fibrin, it is to be expected that TachoSil® will fail in the presence of heparin. Hemopatch is a collagen pad coated with (N-hydroxylsuccinimide functionalized polyethylene glycol (NHS-PEG), which adheres to the tissue, thus better sealing the bleeding. The comparable results in the low and high ranges of efficacy hold promise for suitability of the ex vivo model testing prototypes of new products with a variety of action modes.

Development of new products is inevitably dependent on the testing of prototypes. For medical devices this will, in many cases, mean the execution of animal experiments. In this time of intensified societal debate regarding animal use in healthcare research and the generally adopted 3Rs strategy by researchers and policy makers,¹⁶ the development of an ex vivo model which uses material from a slaughterhouse contributes to the replacement and reduction of live animals for research purposes. The slaughterhouse represents a large source of pig livers. However, in the ethical debate of reducing animal breeding for food supply and the ultimate purpose of the 3Rs to completely avoid using animal (derived) models in healthcare research, our novel ex vivo perfusion model is only a step in the right direction. Further advantage of the model is the limited resources that are needed. An ex vivo perfusion experiment can be executed without the need for an operation room, without professionals performing the anaesthesia and without animal caregivers, thus reducing costs and time. Due to its tabletop design, this setup also offers easy access to every part of the perfused organ, while during in vivo surgery, visibility and access can be more challenging and may need more surgical skills.

This ex vivo perfusion model has a few limitations. The presented model is inherently challenging because of the addition of heparin, which does not favour products that depend on an intact coagulation cascade to accomplish haemostasis. However, when products are working on this challenging model, it is likely that they will work in easier circumstances in the human body. During the prolonged liver perfusion experiments some dark spots were observed on the liver surface, indicating poor perfusion. The poor perfusion was possibly caused by blood clots and may be avoided when the liver is immediately flushed with saline or preservation fluid at harvest, as is done with livers procured for transplantation.^17,18 Standard procedural steps in preparing the animal tissue for food consumption purposes took on average 30 min after cardiac arrest. In this time it was not allowed to open the abdomen and start in situ cold perfusion of the liver. Furthermore, starting cold perfusion in addition to surface cooling after liver harvesting in the slaughterhouse was not possible due to organizational and logistic problems. Thus we decided to only perform surface cooling and accept a prolonged warm ischaemia time with the risk of areas of clotting and impaired perfusion in the liver. Next to these organizational and logistic challenges in the slaughterhouse we did not flush the organs immediately after excision because we found that when perfused with heparinized blood, the severity of standardized bleedings was comparable to in vivo. Despite local thrombosis, sufficient liver perfusion could be obtained in most parts with normal bleeding pattern after injury. Notably, poorly transfused parts of the liver were discarded when lesions were made for the testing of the control products. We used the i-Stat cartridges and hospital blood analysis equipment, validated for human blood and plasma, as porcine related blood analysers were not available in our facility. Similar equipment was used by other research groups showing comparability for most parameters.²¹ Our aPTT measurements gave unrealistic values for the in vivo baseline pigs (all under 20 s, except for one), which is in line with previous findings.²¹ With this model haemostatic products can only be tested for their efficacy in reaching haemostasis. For preclinical development and registration of products, animal experiments are still needed for testing toxicity, biocompatibility and degradation.

Our model is easily adjustable for high pressure arterial perfusion of other organs such as kidneys and spleen. This model could also be useful in understanding pathophysiological pathways and for translational research regarding a range of clinical issues such as thrombosis, nano-medicine and cancer research.

Supplemental Material

sj-pdf-1-lan-10.1177_00236772221138398 - Supplemental material for Validity of a novel ex vivo porcine liver perfusion model for studying haemostatic products

Supplemental material, sj-pdf-1-lan-10.1177_00236772221138398 for Validity of a novel ex vivo porcine liver perfusion model for studying haemostatic products by Edwin A Roozen, Roger MLM Lomme, Nicole UB Calon, Michiel C Warlé and Harry Van Goor in Laboratory Animals

Footnotes

Acknowledgement

M van Erk for her assistance with the schematic figure.

Data availability

The data that support the findings of this study are available from the corresponding author, Edwin Roozen, upon reasonable request.

Declaration of conflicting interests

The author(s) declared no conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship and/or publication of this article.

ORCID iD

Edwin A Roozen

Supplemental material

Supplemental material for this article is available online.

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