GB2347878A - Separating blood using ultrasonic transducer - Google Patents

Abstract

Plasma is clarified from a whole-blood sample B by subjecting it to a radially extending standing wave ultrasound field from an annular ultrasonic transducer 20. A sample tube T containing the blood sample B is located in a recess 12 in a plastics base 10. A cylindrical ultrasonic transducer 20 stands on the base 10 and the annular space between the tube T and the surrounding transducer is filled with water W. The transducer is provided with electrodes over its radially inner and outer surfaces and an ac generator GEN is connected between them. The operating frequency of the generator is selected so that the system is in resonance. The blood cells accumulate at the pressure amplitude maxima of the standing wave field. The clumps of cells then settle out. The transducer may be operated continuously or in pulses. The blood sample may be cooled as the ultrasound field is applied.

Description

CLARIFICATION OF PLASMA FROM BLOOD SAMPLES FIELD OF THE INVENTION The present invention relates to a method of and an apparatus for clarifying plasma from whole blood samples.

BACKGROUND OF THE INVENTION The clinical analysis of plasma samples requires the cells to be removed from whole blood samples. Commonly centrifuges are used for this purpose and provide a highly efficient technique for preparing, as a batch, large numbers of plasma samples. However, where a small number of samples is involved, centrifugation is relatively time consuming because of the manual loading and unloading of samples which it involves. An axial separation technique has been proposed to prepare a small batch of 1 to 10 samples-see Estey, C. A., Felder, R. A. (1996) Clinical Trials of a Novel Centrifugation Method : Axial Separation, Clinical Chemistry 42: 3, pages 402409. Also, a separator is marketed under the trade name PlasmaSep LS by Whatman, which can produce 2ml of plasma from 5ml whole blood using a filter-membrane system: however, the procedure is relatively slow, taking between 30 and 60 minutes.

Blood cell removal has also been demonstrated using ultrasonic standing wave techniques. Thus, when a sample of blood is subjected to a standing wave ultrasound field, the blood cells accumulate at the pressure nodes of the field, at half-wavelength intervals, then sediment out to produce clear plasma-see Baker, N. V. (1972) Segregation and Sedimentation of Red Blood Cells in Ultrasonic Standing Waves, Nature vol 239, pages 398-399. A flow system has also been proposed-see Peterson, S. et al (1986) Development of an Ultrasonic Blood Cell Separator, IEEE Eighth Annual Conference of the Engineering and Medicine and Biology Society, pages 154-156: however, this work succeeded in inferior clarification of the plasma (98.5%) as compared with centrifugation (which reliably achieves 99.9% clarification).

The work with ultrasonic standing wave fields has hitherto involved the use of plane fields, i. e. a planar transducer positioned parallel to a planar reflector. Workers in this field have moreover found it desirable to dilute blood samples prior to exposure to the ultrasonic standing wave field, because the separation efficiency has been shown to decrease significantly for a blood cell concentration of greater than 20% by volume (in human blood, the red cells constitute around 40% by volume).

Yasuda, K. et al, (1997), Using Acoustic Radiation Force as a Concentration Method for Erythrocytes, J. Acoust.

Soc. Am. 102 (1), pages 642-645, investigated the potential damage to erythrocytes caused by acoustic radiation forces when cells were concentrated by a 500kHz standing wave ultrasound field. Using a cell suspension of 3.3% haematocrit in PBS, Yasuda et al concluded that no notable damage occurs under cavitation-free conditions.

It is believed essential to avoid cavitation occurring in blood samples, because the high forces consequently acting on the red cells is likely to cause haemolysis (the breakage of the red cells) with the consequent release of material which might affect assay reliability. It is therefore considered necessary to avoid subjecting blood samples to excessive localised forces, liable to cause cavitation: it has been considered necessary to use relatively high ultrasound frequencies to reduce the likelihood of cavitation occurring.

SUMMARY OF THE INVENTION We have now devised a method of and apparatus for clarifying whole blood samples using a standing wave ultrasound field, which are able to clarify such samples to a degree which approaches centrifugation and in a relatively short period of time, without cavitation.

In accordance with the present invention, there is provided a method of clarifying plasma from a whole blood sample, comprising the steps of providing a whole blood sample in a sample tube, providing an annular ultrasonic transducer, positioning the sample tube so that it is encircled by said annular ultrasonic transducer, and energising said annular transducer to provide a radially-extending standing wave ultrasound field to which the blood sample is subjected.

It has been found that this method is effective in achieving a high degree of clarification of the blood sample, to around 99.8% in under 6 minutes, and even under 4 minutes, in tests which have been conducted. Moreover, it would be expected that because, in the use of an annular transducer, the radial standing wave field is highly focused and concentrated, there would be substantial likelihood of cavitation: however, numerous tests have now been made on whole blood samples, using the method of the present invention, in which no cavitation has been observed.

In some circumstances, we prefer the method to include the step of maintaining the overall system (including the transducer and sample tube of blood) in resonance. Thus, in some circumstances, one operating frequencies is found, within a range of frequencies, at which the voltage measured between the transducer electrodes exhibits a low or minimum value: there is however at least one other operating frequency at which the measured inter-electrode voltage exhibits a minimum (although of higher value than the value of the above-mentioned minimum); we prefer to maintain the system at the latter, or one of the latter, resonant operating frequencies. The resonant frequency may change over time, for example it may change progressively as the blood sample clarifies therefore, preferably means are provided for altering the frequency at which the transducer is driven, in order to maintain resonance.

Preferably a device is connected across the opposed electrodes of the transducer to provide an output signal representing the voltage between those electrodes: this signal is applied to a microprocessor (or a PC) which controls the operating frequency of an ac signal generator which drives the transducer. The microprocessor is programmed to cause the signal generator to effect a tracking step at intervals of time, and monitor the correspondingly-varying output of the voltage detecting device, to determine if there has been any alteration to the frequency at which the measured voltage is a minimum, and if so correspondingly alter the operating frequency, at least until the next tracking step is carried out.

In some circumstances, the above-described voltage minimum is indistinct and therefore difficult to locate and to track. In such cases, tests have been carried out at the adjacent voltage maximum, with substantially equal effectiveness. The frequency of this voltage maximum appears to remain substantially stable over time, so that it may not be necessary to track it: however, the microprocessor may be programmed to track the voltage maximum, in similar manner to that described above, in case this should prove desirable.

Preferably the transducer has a resonant frequency of approximately 1.6MHz (although more generally in the range of 0.7 to 3.5 MHz, preferably in the range of 1.3 to 2.5 MHz).

Tests have found that sedimentation of the red cells occurs more effectively when operating near to 1.6 MHz, than when operating at a frequency of 3.4 MHz or higher; it would have been expected that the use of the higher operating frequencies would be less likely to lead to cavitation, but tests using the method of the present invention have shown that cavitation is avoided at the lower frequencies and, possibly because of the longer wavelengths such that the cells form larger clumps, sedimentation occurs both more rapidly and more effectively.

The ultrasound field may be applied continuously, or it may be modulated. In some circumstances, it may be desirable for the ultrasound field to be pulsed: for example, there may be a plurality of cycles in which the field is alternately"on" and"off" ; however, in the"off"periods, preferably the field remains energised (but at a relatively low energy level) so that the resonance tracking can continue. Preferably the"on" period (duration of each pulse) is longer than the"off"period (interval between successive pulses). Preferably the transducer is energised with a first series of pulses followed by a second series of pulses, the intervals between successive pulses in the second series being greater than the corresponding intervals of the first series.

A preferred transducer has an internal diameter in the range 15 to 35mm. The sample tube may have a diameter typically in the range of 5mm to 15mm. Standard sample tubes of plastics material may be used, for example of polystyrene or of PET.

In accordance with the present invention, an apparatus may be provided for clarifying a plurality of samples (say 10 samples) at the same time. Such an apparatus would have a plurality of tubular transducers, each to receive a sample tube containing a blood sample, each transducer having its own drive signal generator and control arrangement for this signal generator.

BRIEF DESCRIPTION OF THE DRAWINGS An embodiment of the present invention will now be described by way of example only and with reference to the accompanying drawings, in which: FIGURE 1 is a schematic sectional view of an embodiment of apparatus in accordance with the present invention, for clarifying plasma from a sample of whole blood using ultrasound; FIGURE 2 is a schematic diagram of a control arrangement for the signal generator of the apparatus of Figure 1; and FIGURE 3 is a diagram showing a typical relationship between operating frequency and inter-electrode voltage in the apparatus of Figure 1.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT Referring to Figure 1 of the drawings, there is shown an apparatus for clarifying a sample of whole human blood by subjecting it to a standing wave ultrasound field. In the example shown, the apparatus comprises a plastics base member 10 formed with a recess 12: in use, a sample tube T, containing a whole blood sample B, has its bottom end portion seated into the recess 12 to support the tube T vertically. A cylindrical ultrasonic transducer 20 stands on the base member 10, concentric with the sample tube T. The annular space between the sample tube T and the encircling transducer 20 is filled with water W.

In the example shown, the transducer 20 has an internal diameter of 23mm and a length (or height) of 33mm. The sample tube T is formed of plastics material, has an internal diameter of approximately 10mm, a length of approximately 38mm and holds 3ml of whole blood.

The transducer 20 is provided with electrodes over its radially inner and outer surfaces and an ac generator GEN is connected across these electrodes to drive the transducer in its thickness mode. The transducer used has a fundamental resonant frequency at around 1.6MHz. When the transducer is driven, a standing wave ultrasound field is established extending diametrically of the transducer and in all angular directions through the transducer axis A. The standing wave exhibits a primary pressure amplitude maximum on the axis A, and successive maxima at half-wavelength intervals radially outwards from the axis A. In the example shown, approximately 10 such annular maxima are formed within the sample tube T itself.

In use, the standing wave field causes the blood cells to accumulate at the pressure amplitude maxima. The clumps of cells thus formed then sediment out (i. e. fall under gravity): the sample clarifies progressively from the top downwards, leaving clarified plasma at the top of the tube T.

Whilst the ultrasonic transducer 20 has a resonant frequency at about 1.6 MHz, the overall system may exhibit resonance at somewhat different frequencies, each of which may change over time, for example as the blood sample clarifies.

It is possibly desirable to maintain the system in resonance to avoid cavitation: Figure 2 shows schematically a control arrangement for achieving this.

Thus, referring to Figure 2, the ac signal generator GEN is shown connected across the opposite electrodes of the transducer via an amplifier AMP. The operating frequency of the signal generator GEN is controlled by a microprocessor M: a device V is connected across the transducer electrodes to monitor the voltage at this point, and provides a corresponding signal to the microprocessor.

Figure 3 shows a typical variation, with generator frequency, of the voltage across the transducer electrodes.

There is a first frequency fl at which the measured voltage exhibits a minimum V1. However, there are two voltage minima at (f2V2) and (f3V3), either side of a peak P at a frequency (fp) somewhat higher than the frequency fl, although the voltage values at (f2V2) and (f3V3) are higher than at (fla1).

The microprocessor M is programmed firstly to effect a frequency scan, in which it varies the operating frequency of the signal generator progressively over a range of frequencies, continuously monitoring the output signal of the voltage measuring device V, and employs an appropriate algorithm to identify one or other of the two voltage minima (f2V2) and (3V3) : in tests which have been carried out, the frequency corresponding to the lower of these two voltage minima, i. e. the minimum at (fzV2), was identified : it was we have found that the frequency (fl) is inappropriate, because an impedance mismatch appears to arise, whereby relatively little energy is transferred into the standing wave field within the sample tube T. The operating frequencies (f2) or (f3) are substantially more appropriate, even though the measured voltages minima V2 and Vz are higher than V1. Next, the microprocessor M adjusts the operating frequency of the signal generator to the selected resonant frequency (f2) of the system. Thereafter, the microprocessor acts, at intervals of time, to effect a tracking step in which it effects a progressive variation of the operating frequency of the signal generator, over a limited range e. g. a range of the order of 10 kHZ which includes the selected frequency (f2) : during this, the microprocessor M monitors the output from the voltage measuring device V to determine if the frequency, at which resonance is achieved, has altered; if there is any such alteration, the microprocessor runs the signal generator at the new resonant frequency, at least until the next periodic tracking step is carried out.

When using some sample tubes (including polystyrene tubes marketed by Becton-Dickinson under the trade name "Falcon"), the above-described voltage minimum (f2V2) is distinct and can be located and tracked easily. However, with other sample tubes (particularly tubes of PET marketed by Becton-Dickinson under the trade name"vacutainers"), the voltage minimum (f2V2) is indistinct and cannot be located easily or tracked easily: in these circumstances, tests have been carried out, with substantially equal effectiveness, at the frequency (fp), corresponding to the peak P (which is the highest maximum observed in the voltage-frequency characteristic). Moreover, it appears that the frequency of the peak P remains stable over time and may not need to be tracked, although the microprocessor may be programmed to do so if desired.

In tests which have been carried out, the transducer is continuously energised over the duration of the test period.

Instead, the ultrasound field may be modulated. For example, the microprocessor may be programmed to drive the signal generator in a pulsed mode: in particular, the transducer 20 may be driven by the applied signal for a series of 10 cycles each consisting of 10 seconds"on"and 1.5 seconds"off", followed by a series of 15 cycles of 10 seconds"on"and 5 seconds"off", giving an overall duration of 5.7 minutes. The first series of 10 cycles aims to accumulate the blood cells into the annular regions of pressure amplitude maximum: the second series of 15 cycles encourages sedimentation of the clumps of cells in an orderly manner, reducing turbulence caused by cells falling out of suspension. During the"off" intervals, the amplifier gain is reduced to a low level, so that the ultrasound field is maintained at a low energy level, thus enabling the microprocessor to continue tracking the system resonance.

In carrying out tests using the above described method and apparatus, effective clarification of whole blood samples has been achieved, obtaining approximately 1.4ml of plasma from 3ml of blood, the plasma being clarified to around 99.8% in less than 6 minutes, and even under 4 minutes. The plasma may be extracted by dipping a tube into the plasma in the top of the sample tube, after the clarification process.

We believe that it is not necessary for the transducer to extend the full height of the sample tube: instead, the transducer may encircle a short portion of the tube which includes the top of the blood sample; as blood cells are sedimented out of this top portion, plasma rises from the whole blood below and the clarification extends progressively down the sample tube. Alternatively, the sample tube may be displaced upwards as the clarification process proceeds.

It has been found that the temperature of the blood sample rises during its period of subjection to the ultrasound field. The apparatus may therefore include cooling means for preventing or limiting the temperature rise of the blood sample.

Claims (22)

1. CLAIMS 1) A method of clarifying plasma from a whole blood sample, comprising the steps of providing a whole blood sample in a sample tube, providing an annular ultrasonic transducer, positioning the sample tube so that it is encircled by said annular ultrasonic transducer, and energising said annular ultrasonic transducer to provide a radially-extending standing wave ultrasound field to which the blood sample is subjected.
2. 2) A method as claimed in claim 1, in which said annular ultrasonic transducer is provided with two electrodes and ac generator means connected across said electrodes for energising said transducer, and the method comprises the step of operating said ac generator means at a selected frequency.
3. 3) A method as claimed in claim 2, in which said operating frequency is selected such that the system, comprising said transducer, said sample tube and said blood sample, is in resonance.
4. 4) A method as claimed in claim 3, in which said system of said transducer, said sample tube and said blood sample exhibits a voltage-frequency characteristic in which the measured voltage between said electrodes varies with operating frequency, said characteristic having a first minimum at a first frequency and at least one further minimum, the measured voltage at said first minimum being lower than at said further minimum, and said method comprises the step of operating said ac generator means at said further frequency.
5. 5) A method as claimed in claim 4, in which said voltage frequency characteristic exhibits two said further minima, at second and third frequencies respectively, said second frequency being lower than said third frequency, and said method comprises operating said ac generator means at said second frequency.
6. 6) A method as claimed in claim 2, comprising the step of developing an output signal representing the voltage between said electrodes, and responding to said output signal to control the operating frequency of said ac generator means.
7. 7) A method as claimed in claim 6, comprising the step of temporarily altering the operating frequency of said ac generator means at intervals of time, and monitoring the corresponding alteration in said output signal to detect any change in the frequency at which resonance occurs, and altering said operating frequency of said ac generator means to maintain said system in resonance.
8. 8) A method as claimed in claim 2, in which said system of said transducer, said sample tube and said blood sample exhibits a voltage-frequency characteristic in which the measured voltage between said electrodes varies with operating frequency, said characteristic having a first minimum at a first frequency and a maximum at a higher frequency, and said method comprises the step of operating said ac generator at said higher frequency.
9. 9) A method as claimed in claim 1, comprising the step of energising said transducer continuously to subject said blood sample to said ultrasound field for a single continuous period of time.
10. 10) A method as claimed in claim 1, comprising the step of energising said transducer in a pulsed mode to subject said blood sample to successive pulses of said ultrasound field.
11. 11) A method as claimed in claim 10, comprising the step of energising said transducer with a first series of pulses followed by a second series of pulses, the intervals between the successive pulses of the second said series being greater than the intervals between the successive pulses of the first said series.
12. 12) A method as claimed in claim 1, comprising the step of cooling said blood sample during application of said ultrasound field thereto.
13. 13) An apparatus for clarifying plasma from a whole blood sample, the apparatus comprising an annular ultrasonic transducer, means for receiving a sample tube containing a whole blood sample such that said sample tube is encircled by said annular ultrasonic transducer, two electrodes provided on said transducer, ac generator means connected across said electrodes for energising said transducer to provide a radially-extending standing wave ultrasound field to which said blood sample is subjected, and processing means for controlling the frequency at which said ac generator means is operated.
14. 14) An apparatus as claimed in claim 13, in which said processing means controls the operating frequency of said ac generator means such that the system, comprising said transducer, said sample tube and said blood sample, is in resonance.
15. 15) An apparatus as claimed in claim 14, in which said system of said transducer, said sample tube and said blood sample exhibits a voltage-frequency characteristic in which the measured voltage between said electrodes varies with the operating frequency, said characteristic having a first minimum at a first frequency and at least one further minimum, the measured voltage at said first minimum being lower than at said second minimum, said processing means comprising means for operating said ac generator means at said further minimum.
16. 16) An apparatus as claimed in claim 14, comprising means connected across said electrodes to provide an output signal representing the voltage between said electrodes, said processing means being responsive to said output signal to control the operating frequency of said ac generator means.
17. 17) An apparatus as claimed in claim 16, in which said processing means includes means for temporarily altering the operating frequency of said ac generator means at intervals of time, means for monitoring the corresponding alteration in said output signal to detect any change in the frequency of the respective minimum, and means for altering said operating frequency of said ac generator means to maintain said system in resonance at said minimum.
18. 18) An apparatus as claimed in claim 13 in which said processing means includes means for energising said transducer in a pulsed mode.
19. 19) An apparatus as claimed in claim 18, in which said processing means is arranged to energise said transducer with a first series of pulses followed by a second series of pulses, the intervals between the successive pulses of the second said series being greater than the intervals between the successive pulses of the first said series.
20. 20) An apparatus as claimed in claim 13, in which the system comprising said transducer, said sample tube and said blood sample exhibits a voltage-frequency characteristic in which the measured voltage between said electrodes varies with the operating frequency, said characteristic having a first minimum at a first frequency and a maximum at a higher frequency, said processing means comprising means for operating said ac generator means at said higher frequency.
21. 21) An apparatus as claimed in claim 13, in which said transducer has a resonant frequency in the range 1.0 to 2.0 MHz.
22. 22) An apparatus as claimed in claim 13, further comprising cooling means for cooling said blood sample during application of said ultrasound field thereto.