Introduction

Small tubing is used for rodent infusion because of the low dead volume and to match the size of rodent vessels. Restrictions of 0.010in (0.25mm) are common. These restrictions will limit the flow rate at which you can run your infusion studies. In particular, the flow resistance of a tube depends on the fluid viscosity, the length of the tube, and, most importantly, the inner diameter of the tube. If you want to get the same flow rate through a set with narrow openings that you can get through a set with larger openings, you will need to pump at a higher pressure.

Pressures that are too high can wreak havoc with your studies, causing interruptions from unwanted occlusion alarms, undetected leaks from weak joints, or even ruptured infusion sets that spray test article around the room. 

How are you to know that the flow rates proposed for a certain study are appropriate for the infusion equipment you plan to use? You could run a benchtop test with the compound and your equipment to make sure it works, but unless your pumps have modern occlusion alarms, that test is crude. Laboratory syringe pumps can generate dangerously high pressures with no visible signs of trouble.

In this blog we will propose a method of determining a reasonable maximum flow rate range for infusion sets. To start we need to pick a safe maximum range for flow-rate-generated backpressure. What can go wrong as pressure increases? Here is a list:

Infusion timing delayed

With increased flow rates, backpressure will increase causing the infusion lines to expand so that more fluid is stored in the set rather than being delivered to the animal. The tubing expands like a balloon, a plastic syringe itself can expand and the rubber plunger will compress. Softer tubing like polyurethane will expand more than stiffer tubing like polyethylene. In this example we have set a Harvard pump to deliver a bolus of 100µL over 6 seconds (1mL/min) through a standard Instech mouse tether kit, Vascular Access Button™ and 2Fr jugular vein catheter. The pump ran from 0-6 seconds, but the dose was delivered out the end of the catheter between 4 and 25 seconds after the pump was started. Pressure reaches 9 PSI at 1mL/min through this set. Despite the delay, the total volume delivered was accurate, 97.5µL, within expected accuracy limits of plastic syringes (±5%). With longer infusion profiles a startup delay may not matter, but for certain IV boluses, such as those used in IV self-administration, it may be important to understand and manage the delay. In these addiction studies rodents press a lever to get a dose of cocaine or other addictive drug from a pump; the timing is critical.¹

Flow Profile of Bolus Through Mouse Infusion Tether and Catheter²

Pump occlusion alarms triggered

If your pump has one, occlusion alarms are usually tripped between 5 and 20 PSI. The alarm level is adjustable to find the right balance between detecting real occlusions quickly and false alarms, typically from backpressure due to high flow rates and/or a viscous compound. There are plenty of applications where syringe pumps need to operate against high backpressures, like chemical manufacturing, but there should be no reason to exceed 20 PSI when infusing into a live animal. Assuming your pumps don’t need to be serviced, if you are having false alarms either switch to an infusion set with a larger bore or reduce the flow rate, perhaps by increasing the drug concentration.

Undetected leak

As pressures exceed 20 PSI, which can happen if you are using a laboratory pump or pressing down firmly with a manual bolus, you risk failure of the weakest point in your infusion line. Some infusion lines may have joints that can leak under pressure then close back up when the pressure subsides. We know from decades of repairing our fluid swivels that the only application that seems to be able to get them to leak is IV self-administration. Despite the IV set certainly expanding as noted above, for an Instech swivel to leak pressures likely have to have exceeded 60 PSI. (Swivel seals can open temporarily under high pressures, then will reseal as soon as the pressure subsides.) Tubing joints can also leak, as we saw in our 2015 “Impacts of Occlusions on Dose Accuracy” study. These sorts of leaks can easily drip into the bedding and go undetected. At this point your data has been impacted since you have not delivered the full dose. 

Catastrophic rupture

If your infusion line is constructed in a way that resists leaks, at some point the weakest point will rupture in a more dramatic and irreversible way. Tubing can pop off joints, slip fit luers can disconnect, septa in ports can dislodge. The extra fluid stored in the tubing which had been expanded by pressure will spray out. If this happens outside the animal, occasionally it can be repaired, though whatever caused it must be addressed before resuming. If a catheter disconnects inside the animal, the animal is typically lost. If this is happening to you due to flow-related backpressure only and not an occlusion, your rates are far too high for the infusion set you are using.

In this test of an Instech rat infusion set, the catheter disconnected from the VAB™ at a pressure of about 150 PSI before any other joint failed. Note that this is an extremely high pressure; the tether kit which has a resting dead volume of 0.3mL filled to a volume of more than 1mL before it burst. We conducted this failure test with the catheter occluded; the equivalent flow rate to generate this level of backpressure is approximately 20mL/min. 

Pump stalls

Syringe pumps without occlusion alarms typically stall long after the point that an infusion set can leak or rupture. Newer Harvard Apparatus pumps let you select the force limit, a % of maximum force at which the pump will stall. This is NOT an occlusion alarm; it is meant to help prevent damage to syringes, as noted in the manual. If you set this limit too low the pump will not have enough force for normal operation and will stall immediately. As you can see in the table below, none of the pressures at which standard laboratory syringe pumps stall are in the safe range (5-20 PSI) to act as an occlusion alarm, and with small syringes, the pressures these pumps can generate are spectacular.

Pressures Generated Before Stalling³

Flow Rate Limits

Given that occlusion alarms will trip and other bad things can happen when pressures exceed 10 PSI, a safe limit for infusion sets is a flow rate range that generates backpressures of 5-10 PSI. As you can see from this graph of pressure vs flow for a standard rat infusion set, pressure increases quickly once you’ve hit the limit, so stay to the low end of the range if possible to give yourself margin for error.

Pressure vs flow rate in an unoccluded rat infusion set and jugular vein catheter⁴

Continuous Infusion Sets

The table below gives this safe maximum flow rate range for our commonly used mouse and rat continuous infusion equipment. If you are not using these exact part numbers, so long as the flow path is similar the results should hold. If you are pumping a solution that is more viscous than water these limits will be lower.

Manual Dosing

These pressure based limits apply to manual bolus doses as well, though flow resistance will be lower if you are not going through the tether kit. Reaching the maximum safe pressure of 10 PSI takes only 4 ounces of force on the plunger of a 1mL syringe, so it may be hard for you to sense that you are generating unsafe pressures. Note that these bolus rates are based on the limits of the flow path only and are quite high; you may have physiological or animal-welfare-related reasons to go slower. And again if you are infusing a viscous solution the limits will be lower, possibly significantly lower.

Conclusion

If you are not using a data-driven method of assessing whether the flow paths are appropriate for the flow rates of your experiments - perhaps you are doing a few runs at the rate you plan to use and checking that there are no obvious failures - consider these limits based on creating pressures of no more than 5-10 PSI. For most of Instech’s rat VAB™-based continuous infusion sets with 3Fr jugular vein catheters and aqueous solutions this limit is 2-3mL/min. For a similar standard set up with mice it is 0.3-1mL/min.

NOTE: The tests in this blog post were conducted in part by using the real-time pressure readout on the new Instech Syringe Pump.

References

¹Effect of rate of delivery of intravenous cocaine on self-administration in rats, Shindler et al, Pharmacol Biochem Behav. 2009 May 21;93(4):375–381.

²HA1100 pump connected to Instech KVABM1T/25, VABM1B/25, C20PU-MJV2013, primed using pump. Flow vs time measured using electronic balance as described ISO 7886-2 standard.

³Calculated by dividing manufacturers’ stated linear force specifications (35lbs for 11 Elite and NE-1000, 75lbs for PHD Ultra) by syringe cross sectional area (sq.in.), assuming one syringe per pump.

⁴Combination of gravity-based flow tests, pressure readings on the Instech Syringe Pump and extrapolation using pressure vs flow formulas.