Filtration parameters influencing circulating tumor cell enrichment from whole blood

Abstract

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·10(4)∶10(2)∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm(2) track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.

Figure 1. Setup for filtration.

The setup allows for control of either pressure, flow rate or sample dilution factor while monitoring the other two parameters.

Figure 2. Counting cells on a track-etched filter.

False color image of pre-stained culture cells and WBC on a track-etched filter using 4× (A) and 10× (B) magnification.

Figure 3. Pressure versus inverse of open pores, for different flow rates.

The number of pores on a 5 µm track-etched filter was decreased by reducing the number of available filter pores with aperture plates. The fits through the data were used to estimate an average relationship between pressure (P; mbar), flowrate (Q; mL/h) and number of pores (N _open) by fitting the resistance to flow (R); P = µR Q/N _open. With the viscosity µ at 0.9·10⁻³ Pa·s, R is 1.28±0.02 µm⁻³.

Figure 4. Sample flow rate determines pressure drop across filter.

Whole blood was filtered through a 5 µm track-etched filter at total flow rates of 50, 200 and 800 mL/h without sample dilution and with 4 or 16× sample dilution. (A) Total flow rate versus pressure. In contrast with figure 3, the total flow rate is not the main factor contributing to the pressure across the filter when cells are present in the sample. (B) Sample flow rate versus pressure. When the pressure difference is plotted as a function of the sample flow rate, there appears to be a linear relation.

Figure 5. All blood components contribute to total pressure.

Major blood components RBC, WBC and serum were filtered across 5 (panel A) and 8 (panel B) µm track-etched filters until pressure reached a plateau (y-axis). Whiskers show the standard deviation from three measurements. Data-points were all measured at flow rates of 100, 200 and 400 mL/hr, but were slightly offset to facilitate reading of the graph. Inspection of the filters after filtering using bright field imaging and fluorescence imaging of the Hoechst 33342 stain showed no evidence of capture of RBC, while 10³–10⁴ WBC were found on each filter.

Figure 6. Culture cells can pass through small pores at low pressures.

The y-axis shows pressure drops when 1.0·10⁶ SKBR3, PC3-9 or MDA-231 cell lines are filtered across 5 µm track-etched filters with 0.35·10⁶ pores. Data points were all measured at total flow rates of 50, 100, 200 and 400 mL/hr, but were slightly offset to facilitate reading of the graph. The median cell diameter of PC3-9 = 19.0 µm, SKBR-3 cells 16.4 µm and MDA-231 = 15.4 µm and were thus expected to occupy all pores. All samples with SKBR-3 and PC3-9 samples clogged the filter and the sample with MDA-231 clogged at a flow rate of 400 mL/h.

Figure 7. Estimated speeds for different cells.

Estimated speed inside a filter pore versus pressure across the filter for white (WBC) and red blood cells (RBC) and MDA-231 culture cells. Dashed lines are fits of . All three MDA-231 samples at a flow rate of 400 mL/h clogged.

Figure 8. Influence of fixation on recovery, pressure across filter and sample purity.

Bars show recovery of three culture cell lines spiked into 1 mL of whole blood as a function of fixation and pore size (5 µm and 8 µm). Samples were not fixed, fixed for 4 hours in CellSave (CS) or 10 minutes in 0.4% formaldehyde (PFA). Whiskers represent 1 standard deviation. Average sample purity is defined as the % cell line of total cells and is shown below the bar plot. Peak pressure (P) is shown below the bar plot as well. Recovery and purity are both highest for no fixation. The pressure needed to pass 1 mL of blood through the filter is much larger with fixed samples, with the pressure required to pass PFA fixed blood through the 5 µm in excess of 300 mbar that could be determined on our setup.