WO1991005996A1

WO1991005996A1 - Assay method

Info

Publication number: WO1991005996A1
Application number: PCT/EP1990/001723
Authority: WO
Inventors: Per Olof Folkesson Helander; Parvesh Masson
Original assignee: VARILAB AB
Current assignee: VARILAB AB
Priority date: 1989-10-11
Filing date: 1990-10-11
Publication date: 1991-05-02
Anticipated expiration: 1992-04-11
Also published as: GB8922909D0; AU6522490A

Abstract

The erythrocyte sedimentation rate of anticoagulated blood is determined for diagnostic purposes by positioning a sample (2) of the blood in contact with and vertically above or below an essentially horizontally disposed detection surface (12) of an optothermal sensor and permitting erythrocytes in the samples to sediment under gravity away from or towards the detection surface (12), the sample being periodically irradiated through the detection surface (12) with radiation such that heat produced by absorption of the radiation by the sample is detected by a detector (13) and provides signals indicative of the erythrocyte sedimentation rate.

Description

"ASSAY METHOD"

The present invention relates to the measurement of the erythrocyte sedimentation rate in blood samples. The erythrocyte sedimentation rate (ESR) is the most common analysis for monitoring of the acute phase response in human illness. The cellular and biochemical rationale for the increased sedimentation of red cells in disease states is not well understood. Thus, the ESR measurement has been developed empirically through clinical experience over a period of several decades.

The- normal way of carrying out ESR measurement is that (a) blood is anticoagulated with one part of sodium citrate to four parts of blood; (b) the blood sample is either withdrawn in special test tubes or the blood is transferred to a capillary or a teflon tube; (c) the test tube or capillary is positioned vertically and protected from direct light or wind in order to avoid temperature fluctuations; and (d) the test result is read after one hour as the height of the clear plasma zone at the top of the vessel measured in millimeters.

In recent times, various attempts have been made to modify ESR measurement. Since the test is performed in great numbers, quite commonly by general medical practitioners, various devices have been introduced in order to reduce the risk of contamination. Furthermore, various methods for reduction of ESR-time have been introduced. Shortening of the time taken for ESR testing is an important objective for investigation of ESR- ethodology. It has been found that the time can be reduced from one hour to about 30 minutes with the same clinical result provided that sampling tubes of a certain size are used. Recent results indicate that this may be further reduced to about 15 minutes. Other methods which have been tried are centrifugation under various conditions, and in various devices. None of these methods has, however, been successful in the long run. Thus, there is still a need for a more convenient method for carrying out the ESR- test.

Using optothermal spectroscopy, the amount of haemoglobin may be measured directly in a blood sample without lysing the erythrocytes. This is possible because only that part of the sample which is in close proximity to the sensor is measured. The method is not dependent on absorption of light in a diluted sample, and may hence be utilized in very dense samples. ("Principles of optothermal spectroscopy" by P. Helander, Uppsala J Med Sci 9: 155-158, 1986). It has also been observed that erythrocytes sedimenting towards the surface of a photoacoustic sensor cause an increased signal ("Whole blood -a sedimenting sample studied by photoacoustic spectroscopy" by P.Helander and I. Lundstrόm, J. Photoacoustics 1:203-215, 1982), although this was not regarded at that time as a quantifiable phenomenon so that there was no suggestion of any diagnostic applications of the technique.

The present invention is based on the observation that an optothermal sensor is capable of producing a determination of ESR in blood samples in a far shorter time than required by conventional ESR tests and with at least as great accuracy. The method also lends itself to automation in giving an electrical signal from a relatively simple apparatus capable of processing successive blood samples at intervals of only one or two minutes.

According to the present invention we provide a method of diagnosis in which a sample of anticoagulated blood is positioned in contact with and vertically above or below an essentially horizontally disposed detection surface of an optothermal sensor and erythrocytes in the sample are permitted to sediment vertically under gravity away from or towards said detection surface, the sample being periodically irradiated through said detection surface at a wavelength or wavelengths substantially absorbed by the erythrocytes whereby heat produced by absorption of said radiation by the sample is detected and provides signals indicative of the erythrocyte sedimentation rate of the sample.

The optothermal method has many advantages compared to other methods. Since the measurement takes place in close proximity to the detection surface as explained hereinafter, the system is nearly independent of the sample size for all practical purposes.

The reason for a period of one hour for ESR measurements has been claimed to be that sedimentation of erythrocytes goes on in unpredictable time intervals within the hour during which measurements are performed. Our results indicate that a one- or two-minute observation of the sedimentation in the upper 0.05-2 mm (the measuring distance in optothermic spectroscopy) gives the same information. A result which is quantitativly comparable to conventional ESR may be obtained by correcting for acceleration of the particles over the period of measurement and for changes in temperature.

The conventional ESR estimation is a rather rough procedure without temperature standardization, and with an observation method showing gradually decreased precision as ESR increases. The values obtained in the pathological range are therefore quite imprecise, and the method is more or less of qualitative nature. The short period of observation using optothermic spectroscopy may avoid some variations of sedimentation rate due to temperature fluctuations over the conventional one-hour period.

Further our experiments indicate that blood anticoagulated with other agents than those traditionally used for ESR work as well. Thus heparinized or EDTA treated blood may serve for the test. Probably, blood withdrawn in heparinized capillary tubes and processed further in equipment with heparinized surfaces may serve for the measurements. Thus, all blood samples prevented from coagulation may be used according to the invention.

The optothermal sensor may be a thermoacoustic system of the kind described in EP49918. This method is based on a sample cell which consists of a transparent sensitive material connected to piezo-electric crystals and equipped with a light source. When a sample is put on one side of the temperature-sensitive material and exposed to light pulses from the opposite side through the support, the light may be absorbed by the sample. This leads to a temperature increase which in turn expands the support and affects the piezo-electric crystals resulting in a signal which is related to the light absorption. The principle of optothermal spectroscopy may also utilize other types of sample cells. If the piezo- electric crystals are replaced by temperature-sensitive detectors positioned adjacent to the sample the absorbance of light is registered as an increase in heat conducted to such detectors rather than expansion of the material. Such a system is described in WO 90/08952.

Both such systems have in common the use of a transparent detection surface or "window" through which the sample is irradiated by appropriate electromagnetic radiation and into which heat generated in the sample is received to produce an appropriate signal.

Particles suspended in a liquid medium will sediment at a certain rate dependent on, for example, their size, shape and relative weight and the viscosity of the liquid medium. When such a suspension is applied vertically above or below the window of an optothermal sensor and exposed to one or more light pulses, the layer of particles in close proximity to the window will generate a varying signal as the particles sediment towards or away from the window. The absorption of light near the window will increase or decrease as a function of time and the sedimentation properties of the erythrocytes. The optothermal sensor can most conveniently be placed with the sample below the window and with the light coming from above. In this case the signal will decrease as sedimentation proceeds. The optothermal sensor may advantageously be mounted in a flow chamber, whereby the blood sample may initially be agitated or flowing in order to prevent sedimentation. A flow chamber designed to enhance the turbulence of blood flowing therethrough may conveniently be used. Where the optothermal sensor is mounted above the sample, the signal reading under such conditions will be at a maximum and can provide a measurement of the amount of haemoglobin in the sample, for example as described in WO 90/08952. If agitation or flow of the sample is then stopped, the erythrocytes begin to sediment and the signal falls at a rate which provides an indication of the ESR. The method of the invention can thus provide a simultaneous measurement of ESR and haemoglobin level in a single sample, thus avoiding separate determinations.

Periodic irradiation of the sample may, for example, be achieved by irradiating with radiation which is modulated with respect to amplitude and/or wavelength, this being of advantage since it enables background errors such as overall temperature variations largely to be eliminated. Thus the frequency of signal amplification or other periodic means of electronic sampling can be locked onto the modulation frequency of the incident radiation so that extraneous temperature variations occurring between the pulses are not amplified.

Furthermore, the modulation frequency can be related to the rate of conduction of heat from the sample to the sensor. Thus the amplitude of the signals produced by the temperature fluctuations depends in part on the transfer of heat from irradiated sections of the sample at a certain distance from the surface of the transparent solid element. Heat generated at points deeper into the sample is not transferred to the sensor in the time between incidence of the radiation and sampling of the signal from the thermal detector. The maximum depth within the sample from which heat contributes to the signal is termed the "thermal diffusion length" and defines the volume of the sample which is analysed. This definition of the volume makes quantification of an absorbing substance possible. Amplitude modulation of the incident radiation can conveniently be achieved by a conventional mechanical light chopper placed in the collimated light path. Variation of the wavelength of the incident light, e.g. between an absorption maximum and a minimum, may, for example, be effected by a laser diode. In general the modulation frequency should be low, e.g. 2-50 Hz, for example about 16 Hz, more preferably about 8Hz. This gives a relatively short thermal diffusion length, typically of about 0.05 mm in a device of the type described in WO 90/08952, so that the time for the sedimenting erythrocytes to traverse one thermal diffusion length is relatively short, thereby facilitating a short ESR analysis time. On the other hand, for measuring Hgb, it may be preferable for the thermal diffusion length to be longer to provide a more accurate determination which is less affected by sedimentation. This can be achieved by using a lower modulation frequency, for example about 4 Hz. Thus, in assaying both Hgb and ESR in a sample, the initial Hgb determination may be at a lower modulation frequency and the ESR assay may then be continued at a higher frequency. It may often be advantageous to measure both the amplitude and the phase of the optothermal signal output since each of these parameters is affected by and can therefore be used to determine the ESR. Alternatively, especially when using a sensor device above the blood sample, the periodic irradiation may comprise single pulses of radiation at appropriate intervals. Thus an initial pulse of radiation when the sample is homogeneous will give a response at the thermal detector after a delay which is related almost entirely to the conduction time of heat within the sensor. The response signal from the detector accordingly begins shortly after the application of the radiation pulse (which is preferably of extremely short duration) and builds up to a maximum indicative of Hgb absorption. After some sedimentation has taken place and a clear layer has formed between the sensor and the upper layer of erythrocytes, the heat generated by absorption of the pulse has to traverse not only the material of the sensor but also the above-mentioned clear layer. This provides a time delay in the signal from the detector which is characteristic of the thickness of the clear layer and hence of the extent of sedimentation at the time of the pulse. The pulses of radiation can be applied at regular intervals, and are to be distinguished from the amplitude modulated radiation described above, which has far higher frequency, e.g. 4-16 Hz.

Incident light is conveniently led to the sensor by means of an optical fibre system. The light source may, for example, be a laser or a strong lamp. In general, it should be possible to produce incident radiation in the wavelength range 250 nm to 2500 nm.

The thermal detector may, for example, be a thermoelectric device such as thermistor or thermocouple or a thermooptical device such as a temperature responsive laser. The solid heat conductive element may conveniently be made of diamond, which has a heat conductance six time that of copper, or sapphire or quartz, all of which are substantially completely transparent to ultraviolet, visible and infrared light. The solid element is conveniently in the form of a block with two opposed ends and at least one side onto which a thermal detector can be mounted. The sample can then be mounted on or thermally contacted with one of the ends (the "sampling end") while the incident radiation enters the block through the opposite end, the path between the radiation source and the sample thus being unobstructed.

The distance between the sample and the thermal detector is preferably as small as possible, in order to minimise the time for conduction of heat from the sample to the detector and thereby achieve maximum sensitivity. In general, the specific conductivity of the solid element will be many times that of the sample. Typically, the distance of the thermal detector from the end of the element will be of a similar order of magnitude to the dimensions of the sampling end. Thus, for example, the thermal detector might be mounted about 1 mm from a sampling end which itself is about 1 mm across. Alternatively, the surface of the sampling end may extend further along the axis passing through the detector to provide a larger, essentially oblong, area in contact with the sample.

Where the sample absorbs the incident radiation strongly, the latter will readily be absorbed within the thermal diffusion length and produce a strong signal. Where absorption is low, only a part of the incident light may be absorbed within the thermal diffusion length. It will be appreciated that in general, the thickness of the sample should exceed the thermal diffusion length and is preferably at least twice that length.

Sensors used in accordance with the invention will typically be very small. The thermal detector can readily be made of the same size or smaller than the heat conducting solid element. It is particularly convenient to mount the solid element on the end of an optical fibre; the signal from the thermal detector can be conducted by electrical wires or an optical fibre, conveniently mounted parallel to the optical fibre for the incident radiation. Sensors so arranged can be used in a wide range of applications besides measuring ESR, for example not only in in vitro experiments but also in in vivo. Thus, for example, such a sensor may be inserted into a blood vessel for continuous measurement of haemoglobin content.

The thermal detector is advantageously mounted on a surface of the heat conducting solid element which extends parallel to the radiation path. Substantially total internal reflection of the incident radiation at the said parallel surface should then prevent the radiation from reaching the detector. Such internal reflection may be enhanced by attaching the thermal detector to the solid element using an adhesive having a smaller index of refraction than the material of the solid element. Since materials such as sapphire and diamond have a high index of refraction, a wide range of adhesives may be used, including epoxy adhesives, cyanoacrylate adhesives and polyester adhesives. The adhesive may additionally be used to coat the remaining sides of the solid element to minimise egress of light therefrom. Particularly suitable adhesives include electrically conductive glues such as metal epoxy glues, for example a silver epoxy such as Epo-tek H 20 E (manufactured by Epoxy Technology Inc., Mass., USA), since these ensure maximum light retention while also having good thermal and electrical conductivity. Alternatively, the surface of the transparent solid element may be coated with a reflective layer, e.g. a thin layer of aluminium or silver, before attachment of the thermal detector.

It is desirable to shield the sensor from thermal or electrical influences or chemical corrosion. One or more protective layers, e.g. of any appropriate polymer material, may, for example, be applied over the whole sensor, excluding the surface in contact with the sample, in order to achieve this end.

Use of modulated radiation provides a thermal diffusion length which, in general, will be relatively short, e.g. about 0.05-2 mm. The ESR values of normal and pathological blood samples range from 2 to 120 mm per hour. The movement of the erythrocytes through the field determined by the thermal diffusion length is thus very small but the ESR may nevertheless be determined accurately in a remarkably short time as compared with conventional ESR measurements.

Qualitative evaluation may be effected simply by noting the difference in optothermal signal output at two set times after initiation of sedimentation, e.g. at a time 0-20 seconds after initiation and at a time 40-80 seconds after initiation. The two measurements may conveniently be made about 10 seconds and about 60 seconds after initiation. Any observed deficiency in Hgb levels may advantageously be used as a correcting factor to afford an improved correlation in quantitive terms.

Alternatively a series of reading can be taken and used to construct a sedimentation curve (i.e. a plot of signal output against time) , from which an intermediate angle indicative of ESR can be measured.

It is preferable, however, to take at least two sets of readings over the time scale employed, e.g. from a first part and then from a second part of the time scale, which readings can be analysed to give first and second average sedimentation rates from which a standardised erythrocyte sedimentation rage may be determined using any necessary temperature corrections and/or calibration coefficients. In this way it is possible substantially to correct for variations in the sedimentation rate during the observation period (the rate most commonly increases while initially random erythrocyte aggregates reorientate until they reach an orientation permitting maximum sedimentation velocity) .

If the averaged instantaneous rates derived from the two or more sets of readings are identified as R_u R₂.... R_n (which terms should be understood to include parameters such as optothermal signals from which such rates may be derived) a direct correlation exists between conventionally measured sedimentation rates and rates derived in accordance with the invention by multiplying R.,, R-.... R_n each independently raised to empirically determinable positive or negative exponent power. Thus standarised erythrocyte sedimentation rates (ESRs) may be determined by the formula

ESR = C.f(T) . (R^³. (R₂) ^(Rn)

where C is a calibration constant, f(T) is a temperature correction function and each of a, b ... ,z is an empirically derivable positive or negative constant. ,, R₂.... R_n are preferably determined shortly after the onset of sedimentation, e.g. as soon as the signals from the detector employed have stabilised. The rates are advantageously determined within 5 minutes, more preferably within 2 minutes of this onset. One convenient technique is to employ a 90 second measuring cycle in which the detector is allowed to stabilise in the initial 10 seconds, the remainder of the cycle being divided into two 40 second periods in each of which are made a number (conveniently 10) of individual or time- averaged readings. The two series of readings can be processed to generate R₁ and R- by, for example, regression analysis to determine the closest fitting straight line plot of signal against time for each series of readings, the gradients of the two plots corresponding to R₁ and R₂ respectively.

Alternatively the readings or sets of readings may be adapted to one or more polynomial equations of the form

and the desired rate information extracted from the relationship between A, B, C etc.

It will be appreciated that the making, recording and analysis of the readings can readily be put under the control of equipment such as a microprocessor.

Studies of erythrocyte sedimentation rates have shown a substantial temperature dependence of about 10% decrease per °C increase, which can conveniently be quantified in the form

f(T) = α..exp(3/T)

where α is a constant selected to make f(T) equal to 1.0 at normal room temperature (e.g. 22°C) and β is a constant calculated from sample measurements at a range of temperatures T. After an appropriate correction function f(T) has been derived and a statistically significant number of sets of R_]( R-.... R_n values and corresponding ESR values obtained by conventional techniques have been determined it is possible empirically to evaluate the constants a,b....z in the equation

ESR = C.f(T).(R₁)^a. (R₂)^b (R_)^z

so as to generate a substantially linear correlation function which also maximises the correlation coefficient between the R.,, R₂ R_n-derived and the conventionally measured ESR values, the calibration coefficient being chosen so that the regression line has a gradient of as near as possible to 1.0. Again this procedure is conveniently effected by means of a microprocessor. Alternatively, the signals provided by the sensor may be fitted to a polynomial equation as described above and the ESR computed therefrom.

In the accompanying drawings, which serve to illustrate the invention without in any way limiting the same:-

Fig. 1 shows a thermal sensor incorporated into a flow chamber for use in accordance with the invention; and

Figs. 2 and 3 show similar thermal sensors incorporated into alternative forms of flow chambers.

Referring in greater detail to Fig. 1, a block 1 is formed with a flow chamber 2 having an inlet 3 and an outlet 4. A recess 5 in the block 1 is adapted to receive a thermal sensor 6 which rests on an O-ring 7 abutting against a flange 8. The sensor 6 is pressed into contact with the O-ring 7 by springs 9 held in position by a cap 10. The sensor 6 comprises a body of cruciform vertical cross-section provided with a central, vertical, cylindrical hole into which is set a light path 11 leading to a sapphire window 12. A thermistor 13 is provided laterally to the sapphire window 12 and is connected by electrical leads 14 to the signal sensing device (not shown) .

The apparatus of Figs. 2 and 3 is essentialy similar except for the positioning of inlet 3 and outlet 4, which are designed in these embodiments to enhance the turbulence of the blood flow in the region of the optothermal sensor.

The following Examples are given by way of illustration only:- EXAMPLE 1

Blood samples were collected in vacuum test tubes from Becton-Dickenson (length: 120 cm, width: 5 mm) containing citrate solution so that blood was diluted in the ratio one part citrate to four parts blood. ESR values were read after one hour, as routinely done in the laboratory or surgery, to afford conventionally determined values. The samples were thereafter remixed, 1.8ml of each sample was put aside and the residue was fed to the flow chamber of an optothermal spectrometer of the type shown in Fig. 6 of WO 90/08952 using a flow system from AVL (Graz, Austria) at a flow rate of 1 ml per minute. The optothermal sensor used had a sensitive area of 1 mm² and an outer diameter of 3mm and was associated with a 20W halogen lamp the light from which was chopped at a frequency of 16.7 Hz. The flow chamber was rinsed with hypochlorite solution between introduction of each blood sample.

The blood flow from each sample was stopped once the flow chamber had filled, whereupon the erythrocytes began to sediment away from the sensor resulting in a reducing signal. The signal level was noted at 10 seconds and at 60 seconds after stopping of the flow, and the % Hgb reduction over this interval was calculated from the ratio of these signals.

The Hgb-reduction % was plotted against ESR in mm as shown in Fig. 5 of the accompanying drawing; the analysis of the results are related to Fig. 4 as follows:-

All samples with ESR < 30 mm are considered to be normal, whereas ESR > 30 mm indicates disease. If the Hgb-reduction cut off is set so that all samples having ESR < 30 mm are within square a, then, all samples with ESR > 30 mm should appear in area b. False negatives obtained with the optothermic method compared to conventionally determined ESR will appear in area x-

The results shown in Fig. 5 may be summarised as follows:

Area a (normal) Area b (disease) Area x Total

61 47 1 109

Example 2

An optothermal sensor not attached to a flow chamber was used to test the 1.8 ml samples put aside in Example 1. This dip-sensor was mounted at the end of a fibre-optic tubing and was protected from the surroundings by a layer of adhesive covering all surfaces except the 1 x 1 mm sapphire window. Each blood sample was shaken whereafter the sensor was dipped into the sample and positioned vertically throughout the measurements, which were made 10 seconds and 60 seconds after dipping. The results were processed as in Example 1 and are plotted against ESR in Fig. 6 of the accompanying drawings. The results may be summarised as follows:-

Area a (normal) Area b (disease) Area x Total

21 59 2 82

Example 3

The procedure of Example 1 was repeated except that the optothermal signal levels were measured continuously over a period of 60 seconds after stoppage of the blood flow. Fig. 7 of the accompanying drawings is a plot of a sedimentation curve so obtained. The initial disturbances, in part due to delayed stabilisation of the detection equipment, confirm the advantage of allowing a 10 second delay before taking a first reading. The sedimentation angle (s) affords a good indication of the ESR.

Example 4

The procedure of Example 1 was again repeated except that, after a 10 second delay to allow for stabilisation of the signal, signal levels were recorded at 4 second intervals for a total of 80 seconds, the first 10 readings being processed to give an averaged first sedimentation rate R-, and the last 10 readings to give an averaged second sedimentation rate R_. To enhance accuracy the amplitude and the phase of the signals were separately recorded and processed, the former in logarithmic form. Each set of readings was processed to determine the closest fitting straight line plot of signal against time in order to determine the appropriate R₁ and R₂ values.

A total of 111 blood samples were tested. Analysis of the resulting data showed that if in the formula

ESR = C.f(T) . (R^ ) ^~ . (R₂)b

C was assigned the value 984 mm/hour, a the value 2.98 and b the value -1.56 a regression line of gradient 1.02 showing a correlation coefficient between R₁/R₂-derived and conventionally measured sedimentation rates of 0.964 could be constructed. These results are summarised in Fig. 8 of the accompanying drawings, which is a plot of the regression line. It was noteworthy that results for individual samples showed that the ratio R,:R₂ could vary by as much as a factor of two, clearly demonstrating the potential error in sedimentation results derived from a single set of readings which do not compensate for acceleration of the erythrocytes.

It will be apparent from the foregoing that to a useful close approximation erythrocyte sedimentation rates can be determined in accordance with one aspect of the invention by the formula

ESR = C.f(T) . (R,)³. (R₂)^"1-⁵

where C and f(T) are as hereinbefore defined.

Example 5

The procedure of Example 4 was used to determine ESR values for samples of blood anticoagulated with sodium citrate and for corresponding samples anticoagulated with EDTA (1-2% v/v, ca. 0.6 mmol/1) or with heparin (<1% v/v, ca. 20 U/ml) . Correlation curves for the results are shown in Fig. 9 and Fig. 10 of the accompanying drawings and confirm that the results are independent of the nature of the anticoagulant. Thus any blood sample prevented from coagulation may be tested in accordance with the invention.

Claims

1. A method of diagnosis in which a sample of anticoagulated blood is positioned in contact with and vertically above or below an essentially horizontally disposed detection surface of an optothermal sensor and erythrocytes in the sample are permitted to sediment vertically under gravity away from or towards said detection surface, the sample being periodically irradiated through said detection surface at a wavelength or wavelengths substantially absorbed by the erythrocytes whereby heat produced by absorption of said radiation by the sample is detected and provides signals indicative of the erythrocyte sedimentation rate of the sample.

2. A method as claimed in claim 1 wherein the detection surface of the optothermal sensor is situated in a flow chamber.

3. A method as claimed in claim 2 wherein said flow chamber is adapted to enhance turbulence of blood flowing through the chamber in the region of the sensor.

4. A method as claimed in any of the preceding claims wherein the detection surface of the optothermal sensor is above the sample.

5. A method as claimed in any of the preceding claims wherein periodic irradiation of the sample is effected by irradiating the sample with amplitude-modulated radiation.

6. A method as claimed in any of claims 1 to 4 wherein periodic irradiation of the sample is effected by irradiating the sample with radiation modulated such that its wavelength alternates between values corresponding to high and low absorption by erythrocytes.

7. A method as claimed in claim 5 or claim 6 wherein the detector means of the optothermal sensor are sampled at a frequency equal to the modulation frequency of the modulated radiation.

8. A method as claimed in any of claims 5 to 7 wherein the modulation frequency of the modulated radiation is in the range 2-50 Hz.

9. A method as claimed in any of claims 1 to 4 wherein periodic irradiation of the sample is effected by irradiating the sample with short duration pulses of radiation.

10. A method as claimed in any of the preceding claims wherein both signals relating to amplitude and signals relating to phase are observed and/or recorded from the optothermal sensor.

11. A method as claimed in any of the preceding claims wherein the signals from the optothermal sensor are observed and/or recorded at intervals over a time scale not exceeding 5 minutes.

12. A method as claimed in claim 11 wherein the time scale does not exceed 2 minutes.

13. A method as claimed in claim 12 wherein a time scale of about 90 seconds is employed.

14. A method as claimed in any of claims 11 to 13 wherein signals from a first part of the time scale and signals from a second part of the time scale are analysed to give first and second average sedimentation rates, which rates are used to generate a standardised erythrocyte sedimentation rate using any necessary temperature corrections and/or calibration coefficients.

15. A method as claimed in any of claims 11 to 14 wherein individual or averaged signals are observed and/or recorded at approximately 4 second intervals over the time scale employed.