Leukocyte depletion and recovery with Acrodisc™ WBC syringe filter

Cell recovery and viability test

For this test, 20 gamma irradiated and 20 non-gamma irradiated sterile Acrodisc WBC syringe filter units were tested to validate the WBC viability and recovery, as well as to track any changes that the gamma irradiation could cause to the filter device. The samples analyzed were whole human blood samples, collected and preserved using EDTA coated vacutainers. All samples were stored and transported at room temperature; they were vortexed after collection and before the filtration step. All samples were tested within 36 h after collection to prevent WBC viability decreasing below 80%. WBC concentration was determined by flow cytometry before and after filtration of the whole blood samples.

This entire process required:

• Whole blood analysis
• Whole blood filtration: flow cytometry sample preparation
• WBC recovery wash

Testing was performed by the Clinical Flow Cytometry Group from the Department of Blood Sciences at Queen Alexandria Hospital, Portsmouth, UK.

Whole blood analysis

K2EDTA treated whole human blood in samples (Cambridge Bioscience, UK) were analyzed by flow cytometry to determine the initial WBC concentration. For this analysis, 100 µL of the EDTA whole human blood was transferred into a CD45 coated tube (DuraCloneu IM Cell Count Kit, Beckmann Coulter, US). Each tube was vortexed for 30 s and incubated for 15 min at room temperature. After the incubation period, 2 mL of red blood cell lysis buffer (VersaLyseu Beckman Coulter, US) was added to each sample, vortexed for 10 s and incubated for 15 min at room temperature. Finally, WBC concentration was determined by flow cytometry. These obtained values represent the initial WBC count.

Whole blood filtration: Flow cytometry sample preparation

To start the filtration process, the filter was removed from the blister packaging and the upstream side (transparent side with green writing) attached to a 10 mL syringe, from which the syringe plunger had been previously removed (Fig 1.1 and 1.2). Once attached to the syringe, the filter was placed on top of a sterile 50 mL Falcon test tube and secured with tape (Fig 1.3). Prior to filtration, the whole blood sample was mixed by vortexing for 10 to 15 s. Then 5 mL was transferred to the empty syringe barrel to proceed with filtration. The blood was filtered through the Acrodisc WBC syringe filter by gravity and the filtration time was recorded. Once 5 mL of whole blood was filtered, 10 mL of isotonic, flow cytometry compatible phosphate buffered saline (PBS), was added to the syringe barrel and filtered by gravity to wash the membrane of the sterile Acrodisc WBC syringe filter unit. The filtered PBS was collected into the previous Falcon test tubes and the filtrate was labelled as “Leukocyte Depleted Sample” (Fig 1.5).

This sample was analyzed by flow cytometry to determine the WBC loss and viability.

1.1 Remove the sterile Acrodisc WBC syringe filter from its blister packaging.

1.2 Remove the syringe plunger and attach the 10 mL syringe to the filter inlet.

1.3 Place on top of an open 50 mL collection tube and secure with tape.

1.4 Pour the whole blood sample into the syringe barrel and allow the blood to filter under gravity.

1.5 When the blood has filtered through, add 10 mL of PBS solution and filter by gravity.

1.6 The filtrate is the leukocyte-depleted sample.

1.7 The syringe filter is flipped over and the outlet side is connected to a new 10 mL syringe, filled with PBS.

1.8 The filter with the syringe is placed on a new collecting tube, and the PBS is pushed through the filter.

1.9 Multiple backflush steps can be carried out. (3-5 repeats recommended to maximize WBC recovery).

Fig 1. Filtration procedure for WBC separation using the sterile Acrodisc WBC syringe filter.

WBC recovery wash

Leukocytes captured on the filter media were recovered into an elution solution using the backflush procedure. A backflush is a reverse flow setup to elute the WBCs. For this, the syringe barrel was unscrewed, and the sterile filter was flipped over (reversed), as shown in Figure 1.6. Then a syringe was filled with 10 mL of PBS, connected to the white outlet end, and the plunger was pressed down to wash the WBC into a Falcon test tube (see Fig 1.7). As depicted in Figure 1.8, backflush steps using in total 50 mL of PBS (between 3 and 5 syringe refills) were carried out to maximize the leukocyte recovery. The filtrate collected was pooled, vortexed and then labelled as, “WBC Recovery Sample”. Finally, the “WBC Recovery Sample” and the “Filtrate Sample” were ready for further downstream analysis. In this case, samples were prepared for flow cytometry analysis as described in the section, ‘whole blood analysis’. This analysis was to identify the WBC recovery and WBC viability per sample.

Filter overall efficacy (including integrity test)

To evaluate the effect of the gamma irradiation process on the integrity of the sterile Acrodisc WBC syringe filter, an additional 10 gamma irradiated, and 10 non-gamma irradiated filter units were tested. Testing includes a visual inspection, flow rate, bubble point and burst pressure tests, which were designed to verify the device membrane and hardware integrity. All testing was within expected results (see Table 2).

Cell viability and recovery testing

Figure 2 presents the results of the leukocyte depletion from the filtrate sample. These results confirm the ability of the Acrodisc WBC syringe filter to separate the WBC from whole blood samples. Leukodepletion was over 99% in all cases when gamma and non-gamma irradiated syringe filters were used, at both T0 and T2.

Fig 2. Leukocyte depletion (%) values from whole blood samples after filtration with the newly designed sterile Acrodisc WBC syringe filter. Values represent the average calculated from the 20 replicates tested under each condition.

Results from the cell recovery test depicted in Figure 3 show that the sterile Acrodisc WBC syringe filter units were able to recover WBC in the expected range of approximately 60%. Statistically similar values were obtained between the control and the γ-irradiated sterile Acrodisc WBC syringe filter units, and also between the T0 and T2 samples.

Fig 3. WBC % recovery values using the sterile Acrodisc WBC syringe filter; values represent the average calculated from the 20 replicates tested under each condition.

As shown in Figure 4, the viability of the WBCs before (whole blood sample, n) and after the filtration process (WBC recovery sample, n), yielded similar results of approximately 80%. This comparability remained constant between samples filtered with control and γ-irradiated sterile Acrodisc WBC syringe filter units at time zero (T0) and after the 2 yr accelerated aging (T2).

Fig 4. WBC viability (%) from whole blood samples (n) and from the recovery samples after using the sterile Acrodisc WBC syringe filter (n). Values represent the average calculated from the 20 replicates tested under each condition.

Table 1. Results of filtration time, WBC recovery and viability analysis of whole blood samples before and after filtration with non-gamma (control) and gamma irradiated Acrodisc WBC syringe filters.

Note: Cell recovery and viability results correspond to flow cytometry analysis. Values represent the average calculated from the 20 replicates tested under each condition.

Integrity test results

Filter performance pre- and post-gamma irradiation results are detailed in Table 2. All samples met the criteria set out in the integrity testing parameters.

Table 2. Results from the integrity test (IT) performed on gamma and non-gamma irradiated Acrodisc WBC syringe filters at time zero (T0) and two yr accelerated aging (T2). Results from non-gamma irradiated filters were set as a control. Values represent the average calculated from the ten replicates tested under each condition.