Filter tips : A case study on separation performance

This study describes an experimental set-up that allows filter tips to be tested for their separation performance in relation to aerosols. Filter tips are used in air-cushion pipettes to prevent potential contamination of the pipette cones by aerosols. An effective aerosol barrier is essential for preventing false-positive results, especially in highly sensitive PCR experiments. The results show that there are considerable differences between filter tips of different makes with regard to separation performance.

The discovery of PCR has provided molecular biology and medical laboratories with an extremely sensitive method for detecting nuc-leic acids. With the enormous sensitivity that an optimised PCR system can achieve, there is also the risk of generating false-positive results through contamination with nucleic acids. This may be either cross-contamination between different samples or contamination with foreign DNA by laboratory staff or DNA present in the room.

The greatest risk, however, is probably from the PCR product itself, which, produced in large quantities, frequently leads to feedback contamination. It would seem obvious that in the PCR procedure, it is most particularly the steps in which DNA or PCR product is pipetted which present an increased risk of contamination. Cross-contamination occurs if incorrect pipetting causes splashes or drips. Yet even if pipettes are handled properly, aerosols can still form, containing DNA molecules that can thus contaminate the pipette and subsequently different batches. Filter tips are often used to prevent this contamination.

Double-stranded DNA, the main product of PCR, is very long-lived. A primary issue for a PCR diagnostics laboratory therefore has to be the effective avoidance of contamination.

Contamination by aerosols

Most piston-stroke pipettes are based on the air-cushion principle, whereby an air cushion separates the fluid in the tip from the piston inside the pipette. The air cushion is moved by the piston to take up or dispense the fluid in the pipette tip.

The air cushion works like an elastic spring from which the fluid is suspended. When the fluid is taken up through the narrow opening of the pipette tip, aerosols can form as a result of different processes, including, for example, the inadvertent formation of bubbles. The fine mist of fluid formed as a result may contain DNA molecules that get inside the pipette and are transferred to another sample by this route. DNA molecules, present in large numbers and yet very small in size, are most probably contained in an aerosol particle.

However, small PCR amplificates with lengths of around 100 bp are by no means the lower limit for molecules presenting a risk of contamination. Primer molecules with an average length of 30 bp or even individual nucleotides, which, depending on use, may even be marked (radioactively or so as to fluoresce), should be considered the lower limit.

Test system for the separation performance of pipette tip filters

This study tested various filter tips for their separation performance in relation to aerosols. The experiments were performed at the Fraunhofer Institut Toxikologie und Expeimentelle Medizin in Hanover.

Pipette tip filters were tested with regard to their separation performance with aerosols that may possibly form during pipetting. The range of particle sizes of these aerosols can cover several orders of magnitude. The lower range of particle sizes is specified by the size of the molecules typically handled in critical applications (eg, nucleotides). The upper particle size limit is determined by the primary drops arising in the event of incorrect handling (some µm). To perform the test as stringently as possible, the filters were examined in their range of maximum penetration.

Experimental set-up and procedure

The following parameters were selected for performance of the measurements. An NaCl aerosol generated by atomising a 1% solution of NaCl was used as the test aerosol. Figure 1 shows the number-size-distribution of the test aerosol. A condensation particle counter (CPC 3025 from TSI Inc.) was used as the particle counter to individually count the particles passing through the filter. Penetration of the pipette tips was measured at a flow rate of 1 mL/s. This volume flow was determined as a representative mean in tests with experienced users and thus corresponds to actual situations that might be expected in a pipetting procedure.

In the measuring set-up the test aerosol is dispensed into a reservoir, dried and conducted through aerosol neutralizer to a 100mL glass syringe. The syringe serves as a pump that presses the aerosol through the tip. By flushing the syringe several times before each measuring process, it is possible to fill it evenly.

For the determination of the penetration of the filter in the tip an empty tip is compared to a filter tip. A condensation particle counter sucks in the aerosol escaping from the tip at 1 cm3/s at a volume flow of 5 cm3/s. This dilutes the aerosol with air that is kept free of particles by an absolute filter.

This arrangement ensures that all the particles escaping from the pipette tip are measured. Once measurement of the empty tip is complete, the syringe is refilled as described above and the test tip with the filter is measured. The quotient derived from the number of particles which have penetrated the test tip in relation to the number of particles that penetrated the empty tip is the penetration of the filter in the pipette tip:

The separation efficiency of the tip is calculated in accordance with Equation 2.

Results

To determine penetration from the measured values, the ratios of the numbers of particles measured in the test tip to those of the empty tip are first worked out. To do so, the numbers of particles are totalled within a time window with the most constant possible counting rates and divided by one another according to Equation 1. Figure 2 gives a typical curve of counting rates by way of example. The time window typically extends from 23 to 63 s.

In the example selected, the number of particles in the test tip reaches a value of approximately 50 000 particles over the entire measuring period. Three measurements are performed on three tips for each type of test tip. Figure 3 shows the results of the measurements. At 57% of all particles, the tip from Company 1 lets the most through (P = .57, Ed = .43). The tip from Company 2 follows with a penetration of 42%. The tips of Companies 3, 4 and 5 have similar penetration of approx. 11%. Company 6's tip has the best separation performance with the low value of only 3%.

Summary

This study describes a new experimental set-up to allow the separation performance of filter tips to be tested with regard to aerosols. The results show that there are clear differences with regard to the separation performance of filter tips. The quality of the different filter tips with regard to avoiding contamination in methods like PCR can be quantified using the method outlined.

The filter tips from Eppendorf tested had the lowest penetration at 3%, even in the case of the smallest particles, whereas the tips tested from Company 1 allowed approximately half of all particles through. Tests published to date are based on pipetting experiments with highly concentrated DNA solutions, PCR products or radioactively marked nucleotide solutions. Potential contamination of the pipette cone or of the pipette-side part of the filter tip is detected in a subsequent detection reaction (PCR, scintillation counter).

The weak points of these methods are, firstly, that detection is performed indirectly. For PCR detection, the DNA first has to be eluted from the pipette cone, making it difficult to work quantitatively. The sensitivity of the subsequent PCR step is another limiting factor. Furthermore, it is not possible to detect whether aerosols have actually been produced during pipetting. A comparison of different tips is thus not practical using these test methods.

The system presented here individually records every aerosol particle that penetrates the filter. It also works with an aerosol of a defined size and composition. The aerosol generated can be set to be constant and reproducible with regard to particle size, number of particles and flow rate.

Different filter tips can thus be tested reproducibly for their separation performance using a highly sensitive detection method.