AU677001B2 - Apparatus for determining the physical and/or chemical properties of a sample, particularly of blood - Google Patents

Description

OPI DATE 05/10/93 AOJP DATE 09/12/93 APPLN. ID 36429/93 PCT NUMBER PCT/GB93/00475 Il lIll1111l l ll ll H 1111I 1111111 1111111 AU9336429 INTER"', TIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 93/18395 COIN 27/22, 15/05, 22/00 Al A61B 5/05 43) International Publication Date: 16 September 1993 (16.09.93) (21) International Application Number: PCT/GB93/00475 Published With international search report.

(22) International Filing Date: 8 March 1993 (08.03.93) With amended claims and statement.

Priority data: 9205175.4 10 March 1992 (10.03.92) GB (71)72) Applicant and Iuventor: BARNES, Christopher [GB/ GB]; "TAN HWFA", Llanllechid, Bangor, Gwynedd LL57 3LA (GB).

(81) Designated States: AT, AU, BB, BG, BR, CA, CH, CZ, DE, DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SK, UA, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG).

(54) Title: APPARATUS FOR DETERMINING THE PHYSICAL AND/OR CHEMICAL PROPERTIES OF A SAMPLE, PARTICULARLY OF BLOOD (57) Abstract Apparatus and method for correlating certain physical and chemical properties of blood and other samples by means of remote simultaneous multi-frequency dielectric measurement or when one said frequency is applied and compared with an externally entered paramater proportional to the magnitude of a chosen dielectric parameter at another frequency(ies). Apparatus particularly useful for the near instantaneous assessment of the expected sedimentation. condition of red blood cells or other fibrinogen and erythrocyte related parameters.

la Title: "Apparatus for determining the physical and/or chemical properties of a sample, particularly of blood" Description of the invention This invention relates to a non-contacting apparatus and method for investigating certain properties of blood such as red cell count, haemoglobin and fibrinogen content, sedimentation rate and related physical and chemical parameters and for use with similar evaluations in other biological media and for more general use with other samples. In one example the invention is concerned with measurement of fibrinogen to establish instantly the expected sedimentation :o condition of red cells in blood and certain plasma properties. Throughout this embodiment, the term "non-contacting" implies a means remote from the sample and the terms "instant" and "instantaneous" imply very near instant with process times only being limited by the speed of electron flow in circuitry, real time electronic calculation and the operation time of electronic display devices.

Protein concentration in biological media is usually assessed by biochemical methods or by methods of physical chemistry such as viscosity fmeasurement and optical rotational dichroism. Also possible are various forn.

of spectroscopic analysis and chromatography. In one specific situation, that of whole blood, the proteins with the highest concentration are haemoglobin, found in the erythrocyte nuclei and secondly fibrinogen, found dissolved in the plasma.

Fibrinogen concentration is medically important, in that in excess it is a nonspecific indicator of disease state in a person. Fibrinogen levels manifest their effects in a variety of different ways; firstly, they affect the sedimentation rate of the red cells (erythrocytes) giving rise to the so-called erythrocyte sedimentation rate and secondly, they cause effect upon the physical and chemical properties of the plasma.

Manifestations of increased fibrinogen levels have traditionally been monitored in pathology laboratories by two tests, namely; e.s.r. and plasma viscosity mre recently a third biochemical assay, the so-called c-reactive test has also become more popular. E.s.r. tests are however still the most popular with clinicians the world over. The e.s. r test traditionally uses about milli-litres of venous blood and takes one hour to perform, during which time the red cell fraction (haematocrit) separates from the clearer plasma fraction and sediments slowly under the control of gravity and internal viscoelastic forces down a capillary tube or a vacutainer containing preservative, this is very time- •consuming. P.v. and c.r.p are also time-consuming and because in these latter two tests, the red and white blood fractions have to be physically or chemically separate, there if always the chance, albeit remote, that the operatives might become exposed to viral or bacterial biohazard.

Other blood tests such as cell counting and sizing are also carried out *in pathology laboratories using very expensive automated equipment, which needs S' to sample small quantities of blood in close contact by sucking it through a needle type probe inserted by the equipment into a closed vacutainer. Such cell counters, sometimes referred to as haematological analysers, Coulter or similar, are extremely sophisticated 000 S and operate by application of non-linear electrical field gradients and voltage pulses across individual red or white blood cells which have been located by electric or hydrodynamic focusing in a narrow, micron sized, orifice or counting/sizing gate. These machines yield a myriad of parameters, up to 23 in some cases, about the state of nearly all the blood components. Nevertheless they are non-portable and extremely expensive and limited by sample throughput and cleansing procedures. Three of the most important parameters outputted by cell counters are perhaps the red cell concentration the mean cell volume and the haemoglobin content These parameters are considered very useful by many physicia.is in addition to the e.s.r. value in order to make first *a diagnoses and general "state of health" assessments. It is considered useful then by the present inventor if such parameters could be provided by a simpler, cheaper, haematology or haematological analyser technology of greater portability, for use for example in medical practitioners' offices, in the field, or with Third World applications. Of these parameters the problem of haemoglobin has been addressed by previous inventors using optical technology and biochemical lysis of the erythrocytes. However such technology is still quite expensive and because a chemical reaction is involved there is a waiting time before the result is achieved, i.e. the output is not instant.

It is thus an advcantage an erboirment of this invention may provide a means to instantly assess blood fibrinogen levels and their related chemical and physical manifestations remotely and to be able to monitor, also remotely, 6. any or all of the common red cell parameters referred to above, to help preclude biohazard and to provide an analogue or digital readout of all or any of these parameters, in devices that may or may not be configured as a simple form of haematological analyser, and an instant non-optical, non-cell counter means to determine and/or r.b.c. and/or haemoglobin content of blood.

In the case of fibrinogen, it is also an advantage that an embodiment of the present invention may provide an output which can be calibrated in units of concentration, or have units which are effectively dimensionless but whose numerical dynamic range scales and correlates according to any or either of the three common methods of fibrinogen assessment referred to above, or according to a new parameter which the present inventor chooses to refer to as i.s.r. (instant sedimentation rate), but also accounting for and chosen according to the preference of the physician etc.

Automated optical systems have been tried for the assessment of e.s.r.

These are not instant but they do however reduce the time required for a

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measurement down to circa 20 minutes. Methods where the e.s.r tube is spinning in order to increase shear forces on the erythrocytes thereby speeding the rate are also possible.

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"2 'Dielectric methods have also been suggested for the study of timedependent erythrocyte sedimentation, GB 1574681 (Labora Mannheim). In fact, one purpose of this present embodiment is to provide dielectric systems that advantageously and differently however provide for instant (as opposed to timedependent) assessment of e.s.r. value as outlined above.

S* Since various dielectric apparatus has been described in the prior art for making measurements on a variety of samples including even the haematocrit level of blood and in a separate invention, as above, the time dependent sedimentation rate in blood, it is considered very important at this stage in the present embodiment to fully distinguish the prior art from that of this present embodiment. For instance some inventors have described apparatus for making single frequency dielectric and/or conductivity measurements on liquids using two or mor electrodes in direct contact with the sample, either tubular, GB1599241, where using flowing blood in an insulin/glucose control loop, a 500Hz haematocrit level electrode was formed, or annular and four or six in number disposed in alcoholic liquor, at d.c. or very low Sfrequency a.c, GB1460892 (Malcolm-Ellis (Liverpool) Ltd;) these were electrically configured like the standard d.c. four point probe conductivity measurement. This o4* has also been used at a.c. by Kell (US 4965206) in a fermenter, where four pointed contacting probes were used.

The present invention, quite differently, does not function according to four point probe theory or principles, and reiterating, a common disadvantage with the above prior art is that the electrodes actually make physical contact with the liquid under investigation. This can give rise to the chance of electrode fouling, electrolytic effects, and the chance of cross-infecticn and biohazard.

Alternatively alternating voltage of continuously varying (swept) frequency has been applied to a suspension of biological particles, again by means of contacting electrodes, W085/04481 (Public Health Laboratory Service Board).

It is possible that swept frequencies could have been utilised for some aspects of the present invention. However, the degree of signal processing which would have been required was considered unacceptably high by the present inventor and the fact that truly "instant" results would not have been available also dismissed this possibility.

A further disadvantage of the above invention (W085/04481) is that it required separate calibration with its cell full of electrolyte in the absence of biological material in some of its aspects.

This need for separate calibration is precluded in some aspects of this present invention by the use of differential modes as a specific possibility, which advantageously can offset problems of calibration which may be needed due to 1. environmental and temperature effects.

"There have also been a few instances in very simple dielectric measurement and control where a single pair of electrodes have been used on the outside of insulating tubes. One such was a "bang/bang" control device for a drip feed machine, EP ^309085 A2, (Fischer Scientific Co. Pittsburg where capacitor plates were placed either side of a drip feed tube. If the tube became empty or air-locked, the capacitance fell and finding itself arranged in the *feedback loop of a Pierce crystal oscillator circuit), it caused this crystal oscillator to cease oscillating. This type of prior art is adequate for its purpose, i.e. as a warning or on/off control device, but does not have the precision or dynamic range for the applications of this present embodiment.

An example of isolated capacitor plates used for actual measurement purposes with a frequency applied is in the assessment of coal content of fly ash, GB 2 115 933 A (Kajaani Oy (Finland). Essentially such a system worked by monitoring an a.c. level impeded by the combined capacitive reactance of the plates, the insulating tube and the fly ash.

Similarly, and previously referred to above is the method of Labora Mannheim, where two plate or curved electrodes were attached to a test-tube to monitor time-dependent erythrocyte sedimentation, but in this case their effect was in re-tuning a parallel tuned circuit which effectively formed the input tank to a voltage controlled oscillator. A single inductor wound around the tube could even be substituted for the capacitor plates in that invention, being thence *oo* connected in parallel with a separate capacitor and thence to a voltage controlled oscillator Such oscillators are not considered stable enough for use with this present invention. It was also a pre-requisite of the Labora Mannheim system that there should be a measurable distinction between the dielectric constant of the haematocrit and plasma fractions in all cases. Whilst this may be true in most cases, it is the contention and experience by way of experimental observation of the present inventor that at least in the low megahertz frequency band, this is not always the case for certain pathologies at least. It is unfortunate that Labora Mannheim did not specify the operating range of the v.c.o. employed in their description. The above contention may possibly explain why their Sinvention does not appear to have been widely exploited as an e.s.r monitor.

FEB '197 13:59~ GRIFFITH FIRCK A2 r 1 8 612 995VG200 8- Labora Mannheim indica.ted that a single inductor not in contact with the blood could exploit magnetic as well as dielectric properties. Another example of this is EPO1374A9GA2 (Northern Telecom) which used a coil around a' tube coiected to a search oscillator (essentially still a to monitor the flow of mtagnetic particles in a carrier material.

it is clear then that although the prior art indicates some state of the art techniques, these are neither sensitive enough, stable enough, or fast enough.

Thus, it is an advantage that embodiments of the present invention may provide new and more advanced f orms of non-contacting measuring cell, instant methods and apparatus for remote measurement on blood and other fluids based on dielectric principles were unlike the prior art and advantageously to it, there are provided either preferably two or more single, preferably nonvarying stable) frequencies which are simultaneously applied and employed or where if only one such frequency is applied then an external parameter will be requIred to be manually or automatically entered into the calculation circuitry to give a satisfactory resu.lt hitherto not instantly available by other methods of the prior art.

It is a further advantage that the present invention way, provide new kinds of inductive measurement cell and methods and apparatus for applying the above said frequencies, not hitherto described in the prior art.

in a first aspect of the present invention there is provided a method for investigating one or more parameters of a biofluid, comprising the steps of: causing at least two alternating currents at two or more frequencies in the range of 0.5 to 60 MHz to flow simualtaneously through the biofluid; simultaneously detecting a signal corresponding to the magnitude of each said current, respectively; and processing the signals to provide one or more numeric values which correlate with at least one of the S:23487A FEB '97 14:00 GRIFFITH HR(2K 49J5r2 P61 612 99576290 P. 6/16 parameters; aad wherein the currents are caused to f low through the biofluid and the signals are detected without direct contact with the biofluid.

In a second apr-ct of the present invention there is provided apparatus for investigating one or more parameters of a biofluid, comprising: a measurement cell for holding a sample of the biofluid; means for causing at least two alternating currents at two or more frequencies in the range 0.S to 60 M~iz to flow simultaneously through the sample; means for detecting a signal corresponding to the magnitude of each said current, respectively; and is means for processing the signals to provide one or more numeric values which correlate vith at least one of the parameters; and wherein the currents are caused to flow through the sample and the signals are detected without direct contact with the sample, in use, In a third aspect of the present invention there is provided a method for investigating one or more parameters of biofluids, comprising the Steps of; causing at least one alternating current to flow through the biofluid at at least one frequency in the range 0.5 to 60 MI-z Selected to correlate with one of said parameters, detecting a test signal corresponding to the magnitude of each said current, respectively; supply as an external entry a further signal which correlates with another of said parameters; and processing the at least one test signal and the further signal to provide a numreric value which correlates with at least one other of said parameters under investigation; and wherein the at least one current is caused to flow through the biofluid and each corresponding test signal is detected without direct contact with the biofluid.

Accordingly, the present inva~t ion relates to apparatus for determining the physical aud/or chemical 1 properties of a sam~ple, blood or other, with means for retaining the sample, means remote from the sample for applying at least one frequency to the sample, means for measuring the magnitude of the dielectric properties of the sample at each of the said frequencies simultanieously and means for correlating the required physical and/or chemical property of the sample from a simultaneous comparison of the magnitude of a dielectric property of the sample at one of the measuring frequencies with that at the other measuring frequencies or with an alternative parameter proportional thereto.

Furthermore accordingly, the said apparatus of this invention consists of non-contacting dielectric measurement cells linked to electronic circuitry through which external parameters can be entered if necessary.

Accordingly the method involves inserting and retaining samples, blood 0o 9 or other, in the said apparatus, applying the said frequencies, measuring the said o00: magnitudes and correlating the said required physical and/or chemical 9 09 parameters, and providing a scaled readable analogue or digital output by means *of internal electronic (calculating) circuitry. Often one, two or four frequencies are applied.

In the case of two frequencies, one may be between the dielectric alpha and beta dispersions and the other on the high side of the beta dielectric e loss maximum. Whereas in the case of four, all may be on the high side of this beta loss maximum. The method is ideally suited to assessing protein and cellular concentrations in blood and other biofluids but use of other samples is not ruled out. Protein concentration is assessed by its effects on the position and/or magnitude of the high frequency tail of the beta dispersion. Furthermore as a component of some of the measuring cells available to the said apparatus there are provided circumferential electrode structures spaced lengthwise on a former which is electrically insulating and may serve as a tube with one, both or no ends open, into which the separately insulated open or sealed sample tube might be pushed.

Furthermore and advantageously, measurement is made either by monitoring the voltage on the transmitting electrode, the receiving electrode or both, whereas previous inventors have only monitored the voltage on a receiving electrode, or used the capacitance of electrodes to resonate in parallel with an inductor, to tune a v.c.o.

In another aspect of the cells and method, structures as those referred •to above are employed but assessment is of the number density of red cells by measurement at frequencies in kilohertz regions, and assessment of means cell volume in blood is also made with frequencies in the low megahertz regions.

In another form the present invention employs structures as above, but a single frequency is used in conjunction with an electronic circuit which uniquely and advantageously allows temperature compensation and entry of an external parameter such as haemoglobin content from another source such as a cell S* counter or optical haemoglobinometer if the sample is blood, in order to provide S: a more precise output of fibrinogen content, or fibrinogen related parameter(s).

In another form the present apparatus and its non-contacting cells are used for assessing changes in the conductivity and/or dielectric constant of a medium 11 undergoing physical or chemical change e.g. chemical reaction, bioreaction, biochemical or biotechnological reaction, by following the temporal evolution of the eo,'ut parameter.

In a further form the present apparatus is used for any of the uses referred to above but the electrode structnres are replaced by a single coil or inductor wound around and lying in the plane of the former and where the coil via its effect upon a crystal controls the frequency and amplitude of a variable crystal oscillator by series inclusion in the input oscillating tank, not output load and not feedback, circuit. Advantageously such a method is inherently far more stable than those of other inventors that employed free running types of oscillator and not a v.x.o. Since output frequency of a v.x.o. can be measured by a counter very accurately, down to fractions of a Hertz, there is thus concurrent with the increased stability referred to herein an increased precision and o. sensitivity over other methods.

In a further aspect of this present invention, a coil structure surrounds the apparatus measuring cell and the coil (inductor) has low impedance tap or link into which power is fed via a coaxial line from an exciter. Any of the assays and samples mentioned herein may be attempted with the invention configured in this way, since the properties of the sample are mathematically or empirically o: related to the voltage standing wave ratio on the coaxial line as S•measured by a reflectometer or v.s.w.r. meter in that line, provided with or without further d.c. amplification.

0 Yet a further aspect of this present invention is a two frequency measurement cell, method and device, in which a central former is surrounded by four coils or inductors lying coaxially (circumferentially) around it, evenly or not evenly spaced, two of which are non-resonant input (transmit) coils, each sending in a separate single frequency, and two of which receive singly and separately yet simultaneously these original frequencies after passage through the former walls and sample.

In yet a further aspect uf this present invention there is operation as per the electrode based two frequency method described earlier but where a osignal recovery technique is employed on the low frequency receive electrode .o which consists of a high Q ferrite cored inductor connected from the electrode to earth which resonates with the electrode self-capacitance, thereby boosting the fees recovered low frequency signal. This is advantageous because otherwise signals 0 of kilohertz frequencies would suffer very great attenuation after passage through o the high impedance of the former and sample holder walls and would thus be virtually undetectable but for this aspect.

Those skilled in the art will appreciate that other signal recovery methods such as radio frequency amplifiers and/or phase-locked-loop techniques o could also be employed in this context within the scope of the claims of this invention.

00 00 0 o0 S.00 It is an observation of the present inventor that the dielectric (capacitive and conductive) facets of a pathological blood sample measured at individual frequencies in the range 10KHz 1GHz are related to the general state of health of the individual from which the sample was acquired. Thus it is yet one further aspect of this invention to provide means of an electronic general health status indicator based on the observation that there are "norms" of dielectric response at each frequency in the radio frequency continuum and that this can be used with and may contain any of the aforesaid or following aspects of this present embodiment.

It is further asserted by the present inventor that these "norms" arise due to the combined effect of r.b.c, mc.v., Hb, various other proteins, cell e0* membrane leakiness and plasma electrolyte strength upon the loss peak maxima #Goo magnitudes and positions in frequency space of the double or multiple dielectric .41 Beta dispersion of blood, with these dispersive phenomena lying in the approximate frequency range 0.5 60 MHz.

In yet a further form the invention in any of its previous embodiments consists of cells, methods, means and devices capable of measuring without contact and without the use of optics some of the physical dimensions and dielectric properties of sample containers, should these vary from container to container if said containers are filled with fluid of constant chemical and physical composition and dielectric property.

14 In another aspect of this invention, any of the measuring cells methods, and apparatus referred to herein as belonging to this invention may be operated in a differential mode, i.e. using two identical sets of said cells, devices or apparatus with the sample being placed in/measured by one member of said set and a dummy sample, containing for example air, water or electrolyte etc., being placed in/measured by second member of said set. By employing identical components, mechanical and electronic, in each of the said sets and then connecting them to a differential output stage, this aspect of the prese vention allows for the provision of improved results as environmental effects such as temperature will tend to be cancelled by the differential stage.

Although the invention and all its embodiments described herein are primarily illustrated as device(s) for determining protein and cellular "concentration in liquids, preferably whole blood without contact, it is not intended to be limited to the precise detail shown, since various modifications could be made therein within the scope of the claims.

The invention and some of the advantages thereof will now be described more fully by way of reference to the accompanying drawings in which:- *00

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ft FIGURE 1 illustrates the two frequency measurement cell, with its insulating former and outer annular (circumferential) electrode structure for use in this invention; FIGURE 2 illustrates the method of monitoring voltage at the transmit electrode in this invention and shows the stray capacitance path to earth, according to this invention; FIGURE 3 is a diagram illustrating the method of signal recovery, for boosting kilohertz signals after passage through the foimer and sample, according to this invention; FIGURE 4 is a diagram of the alternative measurement cell with an inductor connected to a variable crystal oscillator, for us- with this invention; FIGURE 5 is a diagram of the measurement cell, former, tapped coil and device showing manner of connection to voltage standing wavemeter (reflectometer), according to this invention; *o 0* FIGURE 6 is a diagram of the two frequency four coil measurement cell according to this invention; FIGURE 7 illustrates a block diagram of a two frequency method and device for the measurement of protein, preferably fibrinogen in blood, which device can also be used to measure red cell concentration and/or cell volume by appropriate adjustment

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a:a *O /ST FL' of frequency pairs according to this invention; FIGURE 8 illustrates a four frequency method and device for the measurement of protein, preferably fibrinogen in blood according to this invention; FIGURE 9 illustrates the aspect of this invention where a single frequency device is used in conjunction with an external entry parameter to yield a new parameter, where the entry parameter is preferably haemoglobin content, to yield fibrinogen content or related parameter, at output if sample is blood, and finally, "FIGURE 10 illustrates the differential mode, according to this invention.

Referring to Figure 1 the two frequency measurement cell 10 and 11 are circumferential transmit electrodes remote from the sample, usually, although not exclusively, fabricated from thin brass shim. Frequencies f, and f 2 are simultaneously passed into 10 and 11. 12 and 13 are two similar receiving electrodes from which f, and f 2 are simultaneously recovered. 14 is a central o grounded electrode to minimise stray signal leak along the surface of former/tube 15. 16 and 17 are earthed ground-planes to minimise r.f. radiation from the cell.

Referring to Figure 2, the method of measuring voltage at the transmit electrode, 18 is a crystal controlled oscillator or similar stable exciter.

19 is a 10 picofarad (or thereabouts) trimmer capacitor, 20 is a resistor, usually although not exclusively in the range 5 25 k ohms, 21 is a signal diode. This method has the advantage that detection is made at a relatively high r.f. voltage.

19 and 20 adjust the effective impedance at the transmit electrode to a value which is easily influenced or changed by introduction of a sample tube containing blood or similar into the orifice 22, this change occurs due to leak of signal to earth and the impedance at the transmit electrode being too high to sustain constant current flow, thus the voltage on this electrode will fall when a sample is introduced. Terminals 23 are thence connected to an electronic voltmeter for interpretation.

Referring next to Figure 3, the method of signal recovery for kilohertz frequencies. 24 is a kilohertz frequency generator, presently, though not sete exclusively, a 160 kHz sine-wave and 25 is the receiving electrode whose selfcapacitance 26 brings about high Q resonance with ferrite cored inductor 27 in order to boost the recovered signal appearing for detection at 28.

Reference to Figure 4 shows an alternative measurement cell and single frequency v.x.o. method used with this present invention. Coil 30 is wound around former 29 and is connected in series with crystal 31 to form the input tank circuit of v.x.o. (variable crystal oscillator) 32. The output frequency and a amplitude of 32 will differ a a «i s.

when 29 is empty and when 29 contains a sample in its own tube. They will also differ from sample to sample and will drift if any of the sample properties is temporally unstable. Thus physical and chemical properties of sample may be related to amplitude and frequency of 32. Method is superior to those inventions which have used a v.c.o. due to inherently higher stability of a vx.o. and is superior to those which have used coil in feedback circuit of crystal oscillators for simple on/off bang/bang control.

Referring to Figure 5, shows continuous wave v.s.w.r. method where 33 is an inductor but where an essential feature of which is a low impedance tap S. point or a link. 33 resonates with a capacitor, either self capacitance or inductor and former or external additional parallel capacitance. Power is fed into 33 from *0* exciter 35 via reflectometer or voltage standing wave meter 34. 34 may or may not require d.c. amplification. When sample tube is pushed into orifice of former, resonant frequency of system alters slightly, causing an alteration in the amount of power absorbed by 33 and reflected back towards 35, the change in this reflection or v.s.w.r. is sensed and measured by 34, thus the reading of 34 relates to physical and chemical properties of the sample, within this definition is covered temporal instability of a sample.

*00 0 Reference to Figure 6 shows the inductive variant of Figure 1 a two frequency four coil measurement cell used in this invention. Power is passed in at two frequencies f, and f 2 simultaneously by non-resonant link inductors 37 and 38 respectively. Said frequencies are recovered after passage through the former, sample tube and sample by resonant recovery at parallel tuned circuits 39/41 and 40/42. Any chosen degree of mathematical comparison, calculation or processing then follows on the voltages v 1 and v 2 depending on the precise application and sample type.

Reference to Figure 7 shows a specific use of the invention as a device in block diagram form, preferably for the measurement of fibrinogen in blood.

Frequency f is on the high frequency tail of the dielectric beta dispersion (usually although not exclusively around 50 MHz). 48 and 49, 51 and 52, 47/49 and 58 fees operate exactly as in accordance with the equivalent parts in the voltage monitoring system described in Figure 2.

In the case of blood, the detected voltage is related to the total protein content, being mainly haemoglobin and fibrinogen. Frequency f lies between the alpha and beta dispersions and 45, 46, 50 and 54 operate exactly as in accordance with their equivalent counterparts in the kilohertz frequency recovery method described by reference to Figure 3.

*However, 56 is an extra component which takes the form of a series quartz crystal or similar filter to remove any traces of high frequency signal which may have strayed into this part of the circuit where it is unwanted. The voltage at the detector 57 *Oe

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*S 5 is related to the number density of erythrocytes, if sample is blood, and this number density in turn correlates to a large extent with sample haemoglobin content, for the vast majority of pathological samples (private study of the present inventor).

Said voltage at 57 is also weakly dependent on haemoglobin concentration direct and also on mean cell volume according to a complex mathematical function involving both. Thus appropriate mathematical manipulation of the signals from detectors 57 and 58 in circuit 59 (at its simplest comprising two operational amplifiers) can remove an approximate contribution due to haemoglobin from the total protein function, to leave remaining a signal contribution which depends mainly on fibrinogen levels. The output scale factor may be arranged to yield an output parameter which the present inventor chooses to refer to as the i.s.r. (instant sedimentation rate), if the sample is blood, this parameter may be scaled in magnitude and dynamic range of the more traditional a parameter which physicians are more used to interpreting.

Those skilled in the art however will appreciate that there is no reason why the output should not be scaled in order to give an "instant" p.v. reading or an "instant" c.r.p. reading covering the equivalent dynamic ranges of these two parameters and indeed this is within the scope of the present claims herein.

Referring next to Figure 8, this illustrates a block diagram of the four frequency cell, measurement method and device for use 9 .*0

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5 with this present invention. Because different parts (in frequency space) of the high frequency tail of the dielectric beta dispersion are influenced in different ways by different proteins, e.g. haemoglobin and fibrinogen, if the sample is blood, it is possible to obtain an estimate of fibrinogen levels by simultaneous four frequency dielectric measurement in the frequency range 15 60 MHz (usual but not exclusive range within scope of this present invention). Usually frequency f, is of the order of 17 MHz, f 2 is of the order rf 20 MHz, f 3 is of the order of 30 MHz, and, f 4 is of the order of 50 MHz.

Frequencies f, f 4 are passed in through electrodes 60-63 and out through 65-68 inclusive. 69-72 are narrow band-pass filters centred on f, f4 respectively to assist with signal recovery. 73 is an analogue divider which divides the detected voltage from the 17 MHz filter and detector by that derived for the 20 MHz signal. Likewise, 74 performs a similar operation for f 3 /f 4 For blood as a sample, output functions of 73 and 74 have similar components in respect of haemoglobin but somewhat different for fibrinogen, then weighted subtraction in tends to enhance the effect of fibrinogen and suppress the effect of haemoglobin.

At this point in the circuit the fibrinogen function is almost linear but is superimposed on a d.c. level, thus an appropriate offset is provided by 76 so i. that the output parameter may be displayed at 77. Those skilled in the art will appreciate that the technique is not limited within the scope of the claims to only blood as a sample and indeed any system containing cellular biomass and protein together or even mixtures of proteins will be amenable to this kind of treatment.

When the sample is blood, this aspect of the invention is a most accurate way of determining fibrinogen but because four frequencies are employed, very careful adjustment and initial calibration initially with pathological samples and latterly with electrolyte solutions is necessary and temperature compensation of 73-76 is also desirable. Thus this aspect of the invention is technologically challenging.

Referring next to Figure 9, the block diagram of the aspect of the invention concerned with fibrinogen or protein assessment when a numeric entry parameter haemoglobin) is av.4ilable or known. If haemoglobin content of blood is known or available from another source such as Coulter or similar cell o. counter or biochemical optical haemoglobinometer, and is used as the said external entry parameter then the invention configured according to this aspect can be used to provide a simpler and more accurate assessment of fibrinogen Referring then to the drawing, the main component parts of the system 78-81 operate in exactly the same accord as their equivalent parts indicated in Figure 2.

The digital voltmeter 84 is used with a differential input and temperature is compensated for using potentiometer 82. Those skilled in the art will appreciate automatic compensation also to be possible within the scope of the S. present claims. 83, the haemoglobin entry circuit, is also shown for simplicity as a potentiometer, but in reality in the working demonstrator instrument it comprises of a set of rocker or thumbwheel type 6

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a^ [L \F A switches and it is usually adequate to enter the haemoglobin value to the nearest whole unit. Those skilled in the art will appreciate that there are several other means of haemoglobin entry, both analogue and digital within the scope of the claims of this present invention, including for example acquisition of the haemoglobin level by direct connection to the electronic circuitry of a cell counter or haemoglobinometer.

The action of the system is achieved because the voltage 81 is an inverse function of the total protein content and the differential action of 84 removes from this the haemoglobin contribution and simultaneously allows addition of the temperature compensation voltage. Those skilled in the art will appreciate that the invention configured according to this aspect could be used with multi-component fluid systems other than blood within the scope of the claims of this invention, and that if manually acquired e.s.r. value were available instead of haemoglobin that the system could be configured "in reverse" to yield a haemoglobin value at its output within the scope of these present claims. Those skilled in the art will appreciate that simultaneous frequencies may be applied through just one electrode or inductor, within the scope of the claims of this present invention by using power combiners and/or directional coupling techniques.

Another feature which should not be overlooked when employiig any of the said cells, means, methods and devices referred to in this present embodiment and by way of reference to the drawings is that when the sample is contained in its own container, said container being a tube, vacutainer, capillary etc. with open or sealed end(s), aforesaid container should be a snug push fit into former/tube of said cell etc, Figures 1 9, and there should not be excessive slack or excess air gap (although not all the air is displaced) between this container and the former inner walls.

If the container dimensions vary (from container to container), particularly the internal and external diameters, then errors in the measurement produced by methods and devices herein may arise. Such errors arise from variations in the air gap capacitance where the air gap is that between the said container and former.

It will, however, be appreciated by those skilled in the art that such 0 errors can be reduced/corrected for manually or automatically by tube size correction techniques.

o** Furthermore they will appreciate that this problem may be turned on its head to yield yet a further aspect of the invention referred to above and in the 0 0 claims herein, namely that if samples of fixed chemical and dielectric property are employed in sample containers of nominally the same size but with slight variations in size or dielectric property, then said cells, methods, means and devices may be used to measure a physical dimension of sample container without see• the use of a rule, callipers, micrometer other gauges or optics.

Referring finally to Figure 10, 85 is the sample tube and 87 is the dummy or control sample tube. 86 and 88 are identical formers as illustrated in any of the prior drawings in this present embodiment. 89 and 90 are identical electronic circuits associated with any of the means, methods and devices in this invention. 91 is a difference amplifier and 92 an appropriately scaled output device/display. Effects of temperature and other environmental factors tend to be cancelled by this arrangement, thus making the invention according to this aspect more stable and accurate than those previous disclosures which do not employ a differential mode.

Throughout this embodiment, the sample by way of example has been considered to be on the whole stationary in a closed or open ended sample tube.

Nothing in this embodiment prevents the sample from being a flowing or moving •sample, in which case the formers referred to in every aspect herein would then be of the variety with both ends open. Furthermore, it will be appreciated by oo those skilled in the art, that the aforesaid formers could be fabricated in a "turned inside out" manner i.e. with their electrodes or inductors disposed on the inside and with their ends closed to prevent fluid entry or contact with said electrodes or inductors, thus forining probes which could then be dipped into samples otherwise retained, but yet with operation in accordance with the claims of this present invention. Furthermore, throughout this present embodiment, reference 0000 has been made so far exclusively to non-contacting systems mainly to emphasise *000 their advantages. However, those skilled in the art will appreciate that it is hereby also disclosed that the new methods means devices etc herein can also be made to work when there is contact with the sample material by minimal @00000 modification.

Furthermore, those skilled in the art will appreciate that all the said cells, methods, means and devices referred to herein may be provided with manual or automatic means of sample mixing, handling, labelling etc; and results, analogue or digital, could also be computer stored or on a print-out, and samples may or may not be aspirated from their original containers into second or subsequent containers.

Furthermore, nothing in this present invention prevents the sample from being biomaterial in vivo, small e.g. cells or large e.g. human body digits, limbs etc.

Furthermore, those skilled in the art will appreciate that there is scope S• •for modification in the aspects of the embodiment that refer to simultaneous S •multi-frequency excitation and reception since digital as well as analogue methods S•-0 can be used here and pseudo instantaneous output may be obtained by using fast frequency steps or sweeps of frequencies applied to transmit electrodes.

Furthermore in all aspects where diode detection is employed within this present embodiment, see particularly Figures 2 and 3 and Figures 6 9, this can be replaced by phase sensitive detection as a viable alternative with the dual rconsequence of added sensitivity and two component information from the real and imaginary part analysis, advantageous since in reality samples exhibit complex dielectric behaviour and dielectric constant, sometimes referred to as permittivity 0 0: has such real and imaginary ,arts.

For a said sample dielectric property the present inventor states the real part of permittivity is a measure of the sample a.c. capacitance and with the present invention the apparatus using circumferential electrodes will respond mainly to this capacitive facet, whereas that using coils will respond more strongly to the imaginary part of the permittivity (loss) or conductive facet.

CAT 4 L2

Claims (6)

1. A method for investigating one or more parameters of a biofluid, comprising the steps of: causing at least two alternating currents at two or more frequencies in the range of 0.5 to 60 MHz to flow simultaneously through the biofluid; simultaneously detecting a signal corresponding to the magnitude of each said current, respectively; and processing the signals to provide one or more numeric values which correlate with at least one of the parameters; and wherein the currents are caused to flow through the biofluid and the signals are detected without direct contact with the biofluid.

2. A method as claimed in claim 1, wherein the currents are caused to flow through the biofluid in pairs, the signal detected for one current of each such pair being divided by the signal detected for the other current of the pair.

3. A method as claimed in claim 2, wherein two said pairs of currents are caused to flow through the biofluid, an output from the division of the signals for one of said two pairs being subtracted from an output from the division of the signals for the other of said two pairs to determine a numeric value for one of the parameters of the biofluid.

4. A method as claimed in claim 3, wherein the frequencies are in the high frequency tail of the dielectric beta dispersion of blood, and the parameter under determination is the fibrinogen level of blood. A method as claimed in any one of claims 2 to 4, wherein the frequencies are in the range 15 to 60 MHz.

6. A method as claimed in claim 1, wherein the biofluid is blood and the parameters under investigation comprise any one or more of the following: haewoglobin content, red cell count, mean cell volume, total protein content, fibrinogen level or fibrinogen level scaled in terms of a new parameter referred to herein as the 1U1 FLUI '9f? 140 GIdI 11i I I1 K 99!' Y(ht1 J ',iJi

28- instant sedimentation rate. 7. A method as cl.1aimed in claim 6, wherein the parameter under investigation is the red cell count or a parameter empirically related thereto, and at least one of the frequencies lies between the alpha and beta dielectric dispersions for blood. 8. A method as claimed in claim 6, wherein the parameter under investigation is the total protein content of the biofl-uid and at least one of the frequencies lies in the high frequency tail of the dielectric beta dispersion of blood. 9. A method as claimed in claim 6, wherein the parameter under investigation is fibrinogen level, at least one of the frequencies lies betwet-n the alpha and beta dielectric dispersions of blood so as to provide an output correlating with the red cell count and hence with the haemoglobin level, and at least one other of the frequencies lies on the high frequency tail of the dielectric beta dispersion of blood so as to provide an output correlating with the total protein content, the output correlating with haemoglobin level being subtracted from the output' correlating with the total protein content to produce a numeric value correlating with fibrinogen level. 10, A method as claimned in claim 1, wherein the frequencies are constant. with respect to the value of frequency and amplitude. 11. A method as claimed in claim 10, wherein each of the currents are initiated simultaneously. 12. Apparatus for investigating one or more parameters of a biofluid, comprising: a measurement cell for holding a sample of the biofluidi means for causing at least two alternating currents at two or more frequent.2es in the range 0.5 to 60 M~Hz to flow simultaneously through the sample; memns for detecting a signal corresponding to the iRA magnitude of each said current, respectively; and PLO 14101 GRIF1,1TH Iff-YK (11-i J9Yeh -?[Tl 29 means f or processing the signals to provide one or more numeric values which correlate with at least one of the parameters; and wherein the currents are caused to flow through the sample and the signals are detected without direct contact with the sample, in use. 13. Apparatus according to claim 12, wherein said means for detecting the signals corresponding to the currents are arranged externally of said cell, in use. 14. Apparatus according to claim 12, wherein said means for causing the two or more currents to flow through the sample and the means for detecting the signals corresponding to the currents are disposed within a probe which is inserted into the sample in said cell, in use. Apparatus according to claim 12, wherein the cur~rents are caused to flow and the signals are detected by circumferential capacitive electrodes arranged in line. 16. Apparatus according to claim 12, wherein the currents are caused to flow an~d the signals are detected by multiple inductors arranged in line. 17. Apparatus according to claim 12, wherein the means for causing the currents to flow simultaneously through the sample con~prises a single inductor. 18. Apparatus according to claim 12, wherein each or any of the signals are detected at parallel resonance, in u~se. 19. Apparatus according to claim 12, wherein the signals are detected by frequency selective (filtered) recovery, in use. Apparatus according to claim 12, wherein the signals are detected by phase sensitive detection, in use. 21. Apparatus according to claim 12, wherein any or all of the currents are caused to flow through the sample by an electrode which also detects the signals corresponding to said any or all of the currents, F U 197 J.4!@2 -RIFT 1f),) 6Lc i347(6yfciikd I ,i /lfl) 30 22. A method for investigating one or more parameters of biofluids, comprising the steps of: causing at least one alternating current to flow through the biofluid at at least one frequency in the range 0.5 to 60 MHz selected to correlate with one of said parameters, detecting a test signal corresponding to the magnitude of each said current, respectively; supplying as an external entry a further signal which correlates with another of said parameters; and processing the at least one test signal and the further signal to provide a numeric value which correlates with at least one other of said parameters under investigation; and wherein the .at least one current is caused to flow through the biofluid and each said test signal is detected without direct contact with the biofluid. 23. A method for investigating one or more parameters of biofluids substantially as hereinbefore described with reference to any one of Figures 1 to 24. An apparatus for investigating one or more parameters of biofluids substantially as hereinbefore described with reference to any one of Figures 1 to Dated this 10th day of February 1997 CHRISTOPHER BARNE S By his Patent Attorney GRIFFITH HACK S:23487A