WO2007097702A1

WO2007097702A1 - Non-invasive monitoring of blood flow in deep tissue

Info

Publication number: WO2007097702A1
Application number: PCT/SE2007/050101
Authority: WO
Inventors: Lars-Göran LINDBERG; Jan NÄSLUND
Original assignee: Lindberg Lars-Goeran; Naeslund Jan
Priority date: 2006-02-21
Filing date: 2007-02-21
Publication date: 2007-08-30

Abstract

The invention discloses a method for the generation, detection and evaluation of a photoplethysmographic (PPG) signal to monitor blood characteristics in blood vessels of limited flexibility, such as in vascular compartments and vessels of deep tissues. Underlying the invention are the effects that the orientation and axial migration of red blood cells have on the absorption, scattering and reflection of light (photons) of near infrared and of blue-green wavelengths. In the invention near-infrared wavelength light source (s) and blue-green-wavelength light source (s) are spaced at particular distances from a photodetector (s). This method allows continuous, non invasive monitoring of blood characteristics and changes in these characteristics over time. Data obtained by this method include blood pressure, blood flow, pulsatile blood volume and red blood cell velocity in blood vessels which are rigid or which have a limited flexibility, such as the vascular tissue of bone or in atherosclerotic or stiff vessels.

Description

NON-INVASIVE MONITORING OF BLOOD FLOW IN DEEP TISSUE

FIELD OF THE INVENTION The current invention relates to the field of monitoring and measuring blood volume and blood flow in deep tissues, such as bone and dental pulp but also in muscle and other tissue, non-invasively. Measurements of blood volume, blood pressure and blood flow can be carried out at rest or during functional or physical activities and enable associated conditions and disease status to be assessed, subsequently enabling the need for treatment to be assessed and appropriate therapy regimes to be applied.

BACKGROUND OF THE INVENTION

Basic knowledge on how blood flow is regulated in deep tissue is limited. The importance and interaction of central and peripheral regulation mechanisms are unknown and the etiology of a number of conditions and diseases associated with decreased blood flow are poorly elucidated. Blood flow in deep tissues may be affected by factors acting over a number of hours or even days, with pulsatile blood flow being restricted by mechanical strain, venous occlusion and local changes in pressure in deep tissues, to name but a few examples.

Current methods for studying blood flow and blood volume in deep tissues, such as in bone, dental pulp, large muscles and the like, have certain serious limitations. Few, if any, methods of measuring blood flow are specific for tissue type, with blood flow in tissues such as bone tissue being especially difficult to monitor. Indirect measurements of blood flow in deep tissue, such as bone, have previously been made using bone scintigraphy (BS) (Dye et al., 1993), single-photon emission computed tomography (SPECT) (Gelfer et al., 2003), the ultrasound-Doppler technique (Lustig et al., 2003), the microsphere method (Anetzberger et al., 2004), and positron emission tomography (PET) (lida S et al., 1999). Direct measurements have been made using the Laser-Doppler technique (Hughes et al., 1998) and an intravital microscope television system in combination with confocal laser-scanning optics (Loaiza et al., 2002), but these techniques require surgical manipulation of the bone and may therefore introduce artifacts attributable to local manipulation of the vessels. None of the above-mentioned methods allows the assessment of blood flow continuously and non- invasively in deep tissue during interventions resembling functional activities.

Photoplethysmography (PPG) is a non-invasive optical technique for assessing blood flow-related phenomena which has mainly previously been used to assess blood perfusion in skin (Kamal et al. 1989). It has also been used for measuring muscle blood flow (Sandberg et al., 2005; Zhang et al., 2001). Recently, the technique has also been used to study the effects of sensory stimulation on blood flow in muscle tissue (Sandberg et al., 2004).

SUMMARY OF THE INVENTION

The present invention relates to a novel method for monitoring and measuring blood characteristics, such as blood flow in deep tissue, for example in bone, by establishing that changes in blood flow can be monitored in a rigid blood conduit, or in blood vessels of limited flexibility. Such limited flexibility blood vessels include the vascular system in trabecular bone, vascular tissue in muscle (where one utility of the present invention is the ability to assess changes in blood flow while the subject is in motion) and vascular cells which have lost flexibility, for example in diseases such as arteriosclerosis.

The motility characteristics of red blood cells (RBCs) in circular conduits are exploited by the present invention, in particular the orientation of RBCs and their axial migration and corresponding light absorption and reflection in order to give rise to an AC (alternating current ) and DC (direct current) components of a novel PPG signal. The PPG_Ac signal allows the monitoring of rapid changes in blood characteristics while the PPG_Dc signal allows monitoring of slow variations. Each of the individual signal components can be separated by appropriate filters.

In one embodiment of the invention, a novel photoplethysmographic (PPG) technique is used to assess blood flow in bone tissue. A novel PPG probe is applied non- invasively to the skin overlying the bone containing the vessels to be analysed. The novel probe uses near-infrared and green wavelength light sources (in the case, for example of measurement of blood flow in the patella, wavelengths of 804 nm and 560 nm respectively are used) and the AC component of the PPG signals of the two wave-lengths are used to non-invasively monitor pulsatile blood flow characteristics in the bone tissue and in the overlying skin respectively. For example, blood flow in terms of volume of blood per unit of time, blood pressure (and blood pressure variations) as well as pulsatile blood volume can be monitored. Additional blood characteristics which can be monitored are hematokrit, hemoglobin concentration, oxygen content, oxygen saturation, blood viscosity properties and vessel compliance (wall extensibility). In a further embodiment of the invention the novel PPG technique is used to assess the flow of blood in vessels of soft tissues, for example in vessels such as the radial artery and vessels in the muscle compartment.

In another embodiment of the invention the novel PPG technique is used to monitor and measure the flow of blood in rigid-like vessels, or in vessels of limited flexibility having an inner diameter equal to or greater than 2 mm.

In a further embodiment of the invention the novel PPG technique is used to determine blood flow in rigid vessels, or in vessels of limited flexibility, having an inner diameter of less than 2 mm.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the underside of a photoplethysmography (PPG) probe. The probe comprises in this instance three light sources (one red light-emitting diode and two green light-emitting diodes (LEDs)) and a photodetector (PD). The probe dimensions are 50 mm x 38 mm.

Figure 2 shows a schematic representation of photon (light) distribution during photoplethysmography of the patella. The dark section represents patellar bone, the white area is soft tissue. Abbreviations: photodetector (PD), light-emitting diodes (LEDs) for wavelengths 804 nm and 560 nm.

Figure 3a shows a schematic illustration of the pulsatile PPG (= PPG_Ac) signal. The upper curve trace shows the PPG signal as most often presented on a display or a computer screen. The lower curve trace is the original PPG signal detected with a photodetector placed adjacent to the light source or placed opposite to the light source. Figure 3b shows data mining in order to determine the derived physiological parameters and/or characteristics from the PPG_Ac signal. From the amplitude of the PPG_Ac signal (PPG₃) changes in blood pressure can be derived, from the slope (PPG_Sι_Op_Θ) blood velocity can be derived and from the area under the curve (PPG_auc) the pulsatile blood volume can be derived. The measurement derivations can be rapidly computed and registered.

Figure 4 shows a typical photoplethysmographic recording from bone (804 nm) and skin (560 nm) in one subject at rest. The PPG signals are in arbitrary units. Note the difference in baseline between the two curves; the slow-wave shift at 560 nm may reflect autoregulatorγ activity in the skin whereas such activity is not evident in the recording at 804 nm.

Figure 5 shows the changes in the pulse-synchronous AC component of the photoplethysmographic signal from bone (804 nm) and skin (560 nm) tissues, during different interventions. The results are given as medians, SDs, and ranges, a) Vascular occlusion of skin tissue (n=20); b) Venous occlusion (n=20); c) Arterial occlusion (n=17); d) Application of liniment (n=19).

Figure 6a shows the AC component of a photoplethysmographic signal (PPG_Ac) as a function of the pulsatile blood pressure in a hydraulic model. In the model whole-blood is circulated with a pump where diastolic and pulsatile pressures can be simulated. The PPG signal is obtained in reflexion mode from a rigid, transparent circular tube, where the flow varies in proportion to the pressure (Figure 6b). Mean blood flow (Q) is shown as a function of pulsatile blood pressure. Figure 7 shows a schematic illustration of the RBC distribution in a circular conduit during the systolic and diastolic phase of the cardiac cycle and how the photons are reflected at a theoretical image boundary between the plasma layer and the RBCs.

Figure 8 shows a schematic illustration of the signal generation of the PPG_Ac signal according to a) the blood volume theory and b) the concept of the present invention of migration and orientation of RBCs during the cardiac cycle.

Figure 9 shows comparisons of typical recordings over the radial artery using the novel PPG signal generation and detection of the present invention compared to measurements made on the finger using Portapres for the continuous measurement of blood pressure.

DETAILED DESCRIPTION OF THE INVENTION

Knowledge of regional organ perfusion plays a key role in understanding the physiologic and pathophysiologic processes in various organs. Determinations of actual blood flow in deep tissue such as muscle and skeletal bone are not easily made in humans (Lustig et al., 2003). Different methods measure different levels of the circulatory system, such as limb blood flow and local microcirculation. As mentioned above, blood flow in bone tissue has been previously indirectly measured using bone scintigraphy (BS) (Dye, 1993), single-photon emission computed tomography (SPECT) (Gelfer et al., 2003), Doppler ultrasonography (Lustig et al., 2003), radionuclide-labeled microspheres (Anetzberger et al., 2004), and position emission tomography (PET) (lida et al., 1999). Methods such as Laser- Doppler flowmetry (LDF) (Hughes et al., 1998; Notzli et al., 1989) and intravital microscopy television system in combination with confocal laser-scanning optics have also been applied to attempt more direct measurements (Loaiza et al., 2002). These techniques, however, require surgical manipulation of the bone and therefore may introduce artifacts attributable to local manipulation of the vessels. Near infrared spectroscopy (NIRS) has been used to assess the perfusion index in the tibia bone marrow (Binzoni et al., 2003). To our knowledge, however, no method for measuring local blood flow in human bone tissue continuously and non-invasively has been previously described. Our novel discoveries can also be applied to the measurement and monitoring of blood flow in rigid vessels and in vessels of limited flexibility in soft tissue and offer improved accuracy in terms of measurement results combined with reduced patient discomfort when compared with existing methods and procedures.

The PPG technique in reflection mode requires a light source and a photodetector (PD) placed adjacent to each other. The beam of light is directed toward the part of tissue in which blood flow is to be measured. The emitted light is reflected, absorbed, and scattered within the tissue, and only a small fraction of the emitted light is received by the PD. The intensity of the reflected and scattered light is recorded by the PD and assumed to be related to blood flow changes occurring underneath the probe (Lindberg and ϋberg, 1991). The depth to which light penetrates a tissue is primarily a function of wavelength and the optical geometry of the probe but also of the optical qualities of the tissues of interest. The electrical signal detected by the photoplethysmograph consists of a steady component (DC) — which is related to the relative vascularization of the tissue— and a pulsatile component (AC)- which is synchronous with the pumping action of the heart. The amplitude of the heart-synchronous AC component is thought to be correlated to the blood flow under the probe (Kamal et al., 1989). It has mostly been suggested that the AC component is related to pulsatile volume changes due to varying lumen of the vessel (Challoner, 1979). However, recordings from rigid tissues have shown pulse-synchronous PPG signals (Sakamoto & Kanai, 1979), and the pulsatile component of PPG recordings of the dental pulp has been used to detect blood flow and viability in teeth (Miwa et al., 2002). Moreover, it has been demonstrated in in vitro models that light transmission and reflection in blood can change with the velocity even if the volume of the illuminated blood is constant (Lindberg and Oberg, 1993). Therefore, it has been postulated that both the AC and the DC components in photoplethysmography depend on red blood cell (RBC) orientation and axial migration (Graaf, 1993; Lindberg and Oberg, 1993). Such changes of RBC orientation are known to occur as a function of flow and shear ratio (for review see Fujii, et al. (1999)). It has not previously been described in detail however how the orientation and migration of red blood cells are related to blood flow and shear rate.

We have now discovered that this relationship can form the basis for a new concept for the generation of the PPG signal. This novel PPG signal generation in turn provides new opportunities and possibilities for assessing and elucidating new blood flow related physiological parameters and characteristics. Previously it has been postulated and generally accepted that alterations in blood flow rates occur concomitant with alterations in the physical dimensions of the blood vessels involved i.e. blood vessel narrowing or dilation depending on the volume of blood in the vessel. To date it has been the accepted dogma that the amount of light absorbance directly correlates with such changes in blood volume and consequent vessel diameter. Here we disclose how changes in blood flow can be measured in a rigid blood vessel, or in a vessel of limited flexibility, using a novel PPG signal generation method in which the rate of blood flow (RBC orientation and migration rate), rather than the volume is the determining factor governing absorbance and reflection.

We have discovered that by using at least one light source, emitting light of near- infrared, using a wavelength of from 600-1300 nm, in conjunction with at least one pair of blue-green light-emitting sources, for example LEDs (using a wavelength of from 430-600 nm) and a photo-detector (PD), the monitoring and measurement of blood flow in rigid blood vessels or in vessels of limited flexibility can be carried out continuously, accurately and reproducibly.

In a preferred embodiment of the invention a near-infrared wavelength of 804 nm is used. Thereby, an infrared-emitting light source, suitably a LED is placed 25 mm from a photo-detector (PD), in conjunction with two green light sources emitting light of 560 nm

(placed in a preferred embodiment of the invention 3.5 mm from the same photo-detector) for the monitoring and measurement of blood flow in deep tissue, for example in the patella (Figure 1 and Figure 2). All optical components are embedded in black-coloured silicon and signals from each wavelength are processed in an amplifier and electronically stored on a personal computer. The pulse-by-pulse amplitudes of the AC component of the PPG signal at each wavelength are subsequently extracted with dedicated software (for example, Daquhura 1.3, Linkόpings Tekniska Hόgskola). Mean amplitudes are computed from series of consecutive pulsations (for example 20) before (baseline), during, and after the interventions (for example joint movements) from which measurements are desired.

A typical novel PPG signal is schematically presented in Figure 3a. The lower curve trace is the original one, with a negative deflection corresponding to systole and a positive deflection corresponding to diastole. The upper curve trace is the normally presented signal on e.g. a display, so that the positive deflection (the peak) of the PPG signal or PPG₃ is synchronous with the peak of the pressure curve recorded invasively. An example of how novel parameters can be derived from the PPG signal is illustrated in Figure 3b. A range of different physiological parameters can be derived, based on the new concept for the generation of the PPG signal. PPG₃ is directly proportional to pulsatile pressure variations and can be converted to readings of increase or lowering of blood pressure or systolic pressure in mm Hg. The PPG_Sι_Op_Θ is partially proportional to the maximum velocity of RBCs or rate of blood flow and the pulsatile blood volume can be monitored derived from the data obtained from the area under the curve (PPG_auc)- For a general overview see Figure 3b where;

PPG₃ is proportional to variations in pulsatile pressure and systolic blood pressure; PPGsiope is proportional to variations in maximum pulse velocity; PPGauc is proportional to variations in blood volume of each pulse; PPG_DC is not shown in the figure, but changes slowly over time and is proportional to blood volume.

Blood flow variations as volume per unit of time is proportional to PPG_Sι_Op_Θ multiplied by PPGauc-

In further embodiments of the invention, depending on the tissue to be evaluated and its positioning within the subject, the light source, or multiple sources, emitting light of near-infrared wavelength can be placed within a distance interval of 5 mm to 50 mm of the detector. In a similar manner, the near-infrared light source can be used in conjunction with the light sources emitting green-light wavelengths when these light sources are located within distance intervals of 0 mm to 5 mm of the detector.

In a further embodiment of the invention, for example, blood flow variations are monitored in bone fracture patients for the purposes of diagnostics and prognostics during treatment, utilizing the novel PPG probe to assess blood flow changes before, during and after bone fracture.

In another embodiment of the invention the novel PPG probe is used to assess the effect on blood flow in cases of bone growth in health and disease, in processes where the bone tissue is growing both in healthy subjects and in patients where the bone growth is affected for different reasons. An illustrative example of this utility of the invention is the monitoring of bone growth during physical exercise in athletes during athletics training and competition and during rehabilitation following stress or injury.

In another embodiment of the invention, blood flow characteristics and variations are monitored using the novel PPG probe in cases of hip fracture in elderly patients for screening for selection of treatment modalities. In a further embodiment of the invention, blood flow, blood characteristics and variations are monitored for the purpose of diagnostics and follow-up in osteopenia, osteoporosis (OP) and osteoarthritis (OA) patients before and after surgery.

Yet a further embodiment of the present invention is the assessment of blood flow in atherosclerotic vessels. The novel PPG probe is used to assess the effect of rigid vessel walls on blood flow by assessing the difference between blood flow related parameters in normal vessels and in atherosclerotic vessels.

Another embodiment of the present invention is the use of the novel PPG probe in the assessment of blood flow in connective tissues.

In summary, the present invention can be used to compare the pattern of pulsatile blood flow in individual patients before and after an intervention, or analysis of movement. The present invention can also be used to assess possible differences between patients and controls in a variety of deep tissues without knowing the rate of blood flow, which enables the invention to be used in continuing research into the underlying characteristics of various diseases, conditions and mechanical injury. The importance of using a non-invasive instrument for measuring blood flow in a variety of instances is clear and is exemplified by a number of studies on knee surgery where disruption of the vessels supplying the patella, for example, has been shown to occur after lateral release and anterior cruciate ligament reconstruction with bone-tendon-bone autografts (Bonutti et al; 1998).

The following examples are intended to more fully illustrate the invention and are not intended to limit the invention in any way whatsoever. One of skill in the art will recognise, from the teachings outlined herein, that, without deviating significantly from the spirit of the invention, a number of modifications may be introduced and that such modifications are intended to be encompassed within the scope of the invention.

EXAMPLES EXAMPLE 1.

Estimation of blood flow in human patellar bone.

Twenty healthy normotensive subjects with no history of knee pain were recruited. All subjects, members of a health club, were used to physical activity. All PPG recordings were made on one occasion and all subjects gave their informed consent to participate. The research ethics committee at the Faculty of Medicine, University of Lund, approved the study.

1.1 Measurements.

The subjects lay in a supine position in a quiet room with moderate light and a room temperature of 23 ⁰C (±1°C). The novel PPG probe (see Figure 1) was placed over the centre of the patella bone and attached to the skin with adhesive tape. After 15 min of rest, blood flow was recorded continuously from 60 s before the intervention to 5 min after. Blood flow was measured with the knee fixed (by means of a vacuum pillow, AB Germa, Sweden) in a position at 20° of flexion during all interventions. The PPG signal was analyzed by an investigator who was blinded to the subjects and recording conditions. Various procedures were used to influence the blood flow superficially in the skin or in the patella bone. The nature and purpose of the interventions are described in more detail below.

1.2 Vascular occlusion of skin tissue. After the initial 60 s of measurement to establish a reference value, a pressure of

100 mm Hg was applied to the probe with an algometer (Somedic®) to occlude the skin blood flow. The pressure was applied for 60 s, after which the pressure was removed. The purpose of this experiment was to investigate the ability of the PPG instrument to differentiate between skin and bone blood flow when the blood flow in the skin was occluded. For results see section 1.6 below and Figure 5a.

1.3 Venous occlusion.

After the initial 60 s of measurement to establish a reference value, a pressure of 60 mm Hg was applied around the thigh with a blood-pressure cuff to decrease the venous blood flow (Groothius et al., 2003). The cuff remained inflated for 60 s, after which the pressure was released. The purpose of this experiment was to investigate how the PPG signal responds to partially decreased blood flow in tissue segments upstream to venous occlusion. For results see section 1.6 below and Figure 5b.

1.4 Arterial occlusion.

After the initial 60 s of measurement to establish a reference value, a pressure of 180 mm Hg was applied around the thigh with a blood-pressure cuff to block arterial and venous blood flow. The cuff remained inflated for 60 s, after which the pressure was released, and the recording was continued for 5 min. The purpose of this experiment was to investigate the response of the PPG instrument to arrested blood flow and to detect possible differences between blood flows in the skin and bone tissue during post-occlusion reactive hyperemia. For results see section 1.6 below and Figure 5c. 1.5 Application of liniment.

Liniment (Transvasin®) was applied to the surface of the skin over the patella to increase skin blood flow by vasodilator activity (Fulton et al., 1959). The active substances in Transvasin® (tetrahydrofurfursalicylate, ethyl nicotinate and hexylnicotinate) increase skin perfusion, and nicotinic acid has a dilating effect after a few minutes (Sandberg et al., 2004). PPG recordings were performed for 60 s to establish a reference value. The probe was then moved, and a minor amount of liniment was quickly applied to the surface of the PD. After replacing the probe at the same site on the patella, blood flow was measured for another 5 min. The purpose of this experiment was to investigate the ability of the PPG instrument to differentiate between skin and bone blood flow when a substance known to induce skin vasodilatation was applied to the skin. For results see section 1.6 below and Figure 5d.

1.6 Statistical Analysis. The statistical package Statistica 7.1 (StatSoft, Inc., USA) was used for statistical analysis of the data obtained in sections 1.1 - 1.5 above. Mean values and standard deviations (SDs) were calculated for the anthropometric data. Differences in blood flow between individual pairs, based on assessments before and after intervention, are expressed as percent of pre-intervention control, and the results are given as medians, SDs, and ranges. Wilcoxon's paired signed rank test was used to test for differences between blood flow in skin and bone tissue. The level of significance was set at P < 0.05. In the physical model (section 1.6 above) the correlation coefficient was determined using a computer program (MATLAB 7.0) to assess the association between the PPG signal and the blood pressure. Figure 4 shows a typical PPG recording from bone (804nm) and skin (560nm) at rest in one subject. There was a slow variation in baseline of the skin PPG signal but not in the corresponding signal from bone tissue. The amplitudes of the AC component during the interventions are presented in Figure 5a-d. Values are expressed as percent of pre- intervention control. In the results, the mean values from the left and right knees are presented. Some recordings of the PPG signal were not analyzed due to 50 Hz noise and in such instances only the recordings from one knee were analysed. In some interventions, fewer than 20 subjects were analyzed, also because of high noise in the PPG signal.

Vascular occlusion of skin tissue is shown in Figure 5a. The PPG signal from the skin over the patella (560 nm) was blocked after application of a local pressure of 100 mm Hg. The signal from the patellar bone (804 nm) showed some influences from the skin occlusion but was not significantly decreased (P=O.63). There was a significant difference in the signals from the tissues (PO.001). Venous occlusion is shown in Figure 5b. During venous occlusion, a significant decrease in both signals was found (bone, P < 0.001 ; skin, P = 0.001). The signal from the skin decreased more than the signal from the bone (P = 0.052).

Arterial occlusion is shown in Figure 5c. The PPG signals from the skin and the patella were absent during arterial occlusion. After the pressure was released, a significant increase, compared with baseline, was seen in the signal from the patella (P < 0.001) but not from the skin (P = 0.54), and the difference between the signals of the two tissues was significant (P = 0.02).

Application of liniment is shown in Figure 5d. After liniment was applied, the PPG signal from the skin increased significantly (P < 0.001) while the increase in the signal from the bone was non-significant (P = 0.07). There was a significant difference between the signals of the two tissues (P < 0.001).

EXAMPLE 2. In vitro study of the PPG signal in a rigid tube.

The rigid flow-through model consisted of a hole (diameter 2 mm) drilled in a piece of acrylic glass (PMMA). In the reflection mode of photoplethysmography an optical detecting fibre (diameter=1 mm) was placed adjacent to an illuminating fibre (diameter=1 mm) with a centre to centre distance of 2.5 mm and a distance of 2 mm between the fibres and the hole. A LED (wavelength 880 nm) was used as the light source for the illuminating fibre. The light from the detecting fibre was guided to a silicon PD (CERLED, Germany). The measurements were performed on blood from 12 healthy blood donors, with Hb concentrations ranging from 116-162 g/l. The blood was circulating in a silicon tubing system described earlier (Lindberg and Oberg, 1993). A waveform generator regulated a roller pump (Mekaneljo, Sweden), which produced a simulated pressure waveform closely resembling the human pulsatile blood pressure (Lindberg and Oberg, 1993; Borgstrόm, 1981). Blood flow in ml/min was determined by collecting the blood for 60s at each pressure level. Measurements were made with both whole blood and hemolyzed blood.

Figure 6a shows relative changes in the PPG AC signal when the pulsatile pressure was varied between 0 and 100 mm Hg, superimposed on a constant diastolic pressure of 70 mm Hg and at a constant frequency of 1 Hz (corresponding to 60 beats/min). Figure 6b shows that the blood flow (ml/min) through the rigid tube varied linearly with the intra-tube pressure. The experiments were performed using both whole- and hemolyzed blood from the same donors. With whole blood the PPG AC-signal varied with the pressure pulse. Plotting the values from PPG AC-recordings against the pulsatile pressure, the correlation coefficient was 0.82. With hemolyzed blood no pulsatile signals were recorded. EXAMPLE 3:

Decreased pulsatile blood flow in the patella in the patellofemoral pain syndrome.

Using the novel PPG probe, we examined the role of ischemia in the patellofemoralpain syndrome (PFPS). We hypothesized that flexing the knee joint would interfere with the perfusion of the patellar bone in PFPS. Pulsatile blood flow was measured continuously and noninvasively using the novel photoplethysmography probe. Measurements were made in a resting position with a knee flexion of 20° and after passive knee flexion to 90°. In total, 22 patients with PFPS were examined bilaterally and 33 subjects with healthy knees served as controls. The pulsatile blood flow in PFPS patients was found to decrease during passive knee flexion from 20° to 90° (95% Cl for relative position [RP] = -0.48 to -0.17) while the response in the control group showed no distinct pattern (95% Cl for RP = -0.05 to 0.31). The difference between the groups was significant (P=0.0002). We conclude that pulsatile blood flow in PFPS patients is markedly reduced when the knee is being flexed, which supports the notion of an ischemic mechanism in PFPS.

EXAMPLE 4:

Generation of the novel PPG signal of the present invention in rigid blood vessels.

The novel PPG signal generation of the present invention is schematically illustrated in Figure 7 by the relationship between the core of red blood cells (RBCs) and the majority of the plasma layer in a cross section of a circular rigid tube (no pulsatile volume changes because there is no variation in the lumen/diameter of the tube (vessel)). To the left more RBCs are accumulated or migrate towards the axis of the tube (which corresponds to the systolic higher pressure) and to the right the RBCs are allowed to be distributed towards the periphery of the tube (corresponding to the diastolic lower pressure in the tube/vessel). In the systolic phase the theoretical image boundary plasma layer is thicker and in the diastolic phase it is thinner.

Figure 7 also shows schematically the photons (=light) reflected at the theoretical image boundary between the plasma layer and RBCs as a result of the differences in refractive index in combination with both the distance between the wall and the core of RBCs and the wavelength of the light used, here near-infrared light (780-1200 nm). Light reflection in plasma is also lower compared to reflection in solute containing RBCs. During systole the increased distance and the thicker plasma layer reduces the amount of light reflected back towards a photodetector and during diastole the decreased distance and the thinner plasma layer increases the light reflected back. All this taken together explains the origin and the phase of the PPG_Ac signal shown diagramatically in the upper trace curves of the figure. EXAMPLE 5:

Comparison of mechanisms underlying PPG^n signal generation The differences between the mechanisms generating the PPG_Ac signal are illustrated schematically in Figure 8. In Figure δa) it is assumed that pulsatile blood volume changes followed by pulsatile light absorption gives rise to the PPG reflection mode signal synchronous with the pulsatile pressure. In b) and according to the disclosures of the present invention the pulsatile PPG signal is generated by different light reflection and absorption due to migration/orientation and relaxation of RBCs during the systolic and diastolic phases, respectively. This does not require pulsatile blood volume variations or diameter changes of the tube/vessel.

EXAMPLE 6:

Recordings over the radial artery of a subject using PPG, upper curve, and blood pressure measured on a finger using a Portapres commercial instrument, lower curve, in a six second interval in a patient at rest are illustrated in Figure 9. PPGAC and Portapres blood pressure units are in volts (V). There is a strong resemblance in pulse to pulse comparison between PPG and Portapres blood pressure corresponding to a correlation coefficient r = 0.95. Similar PPG recordings over the radial artery to those shown in Figure 9 have been obtained for different applications, such as in patients undergoing a dialysis session of 4-5 hours and in patients under neurological intensive care. Recordings in accordance with Figure 9 over a vessel of diameter approximately < 2mm resembling a rigid vessel may be performed on a variety of vascular beds and for different applications. The degree of atherosclerosis of a vessel, for example, may be determined by frequency analysis of the PPG signal based on our novel concept where the orientation/migration of RBCs are distorted in a specific way. Similar signal analysis may be used for determination of stiff vascular walls.

EXAMPLE 7:

Hemodynamic instability is a common complication during dialysis treatment and has been postulated to result from intravascular fluid loss and the inability of the cardiovascular system to compensate for this, leading in turn to hypotension and reduction in the effectiveness of dialysis treatment. There is currently no effective or accurate monitoring system available to the clinician to assess the degree or extent of hemodynamic instability in the individual patient. Using the PPG signal generation and detection method of the present invention we can measure and periodically or continuously monitor the hemodynamic status of an individual before, during and after dialysis, both using direct PPG signals and derived measurements. In the measurements and monitoring of dialysis patients two monitoring devices are used; one placed over the radial artery and one placed over the muscles of the underarm.

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Claims

1. A method of non-invasive measurement and monitoring of blood characteristics comprising: a) generating a photoplethysmographic signal using at least one near-infrared light source, in conjunction with at least one pair of blue-green light sources and a photo-detector; b) detecting an AC component in the photoplethysmographic signal; and c) correlating the AC component to a blood characteristic or to a change in a blood characteristic.

2. Method according to claim 1 wherein the near-infrared light source emits of a wavelength within an interval of 600-1300 nm and the light emitted by the blue-green light sources emits light of a wavelength within an interval of 430-600 nm.

3. Method according to claim 1 wherein the near-infrared light emission is of a wavelength of 804 nm and the light emitted by the blue-green light sources is of a wavelength of 560 nm.

4. Method according to any of the preceding claims wherein the near-infrared light source is placed within a distance of 5 mm to 50 mm of the detector and wherein the blue- green light sources are placed at a distance of between 0 mm to 5 mm of the detector.

5. Method according to claim 3 wherein the blood characteristic is measured in deep tissue.

6. Method according to claim 5 wherein the deep tissue is bone.

7. Method according to claim 6 wherein monitoring of blood characteristics is carried out to assess blood flow changes before, during and after bone fracture, such as hip fracture screening, for examination, diagnosis and follow up in patients with osteopenia, osteoporosis or osteoarthritis, before and after surgery.

8. Method according to any of the preceding claims wherein monitoring of blood characteristics is carried out to assess sports-related or stress-related injuries.

9. Method according to any of claims 1 to 4 for the assessment of blood flow in rigid blood vessels or in vessels of limited flexibility such as atherosclerotic blood vessels.

10. Method according to any of claims 1 to 4 for the assessment of blood flow in connective tissues.

11. Method according to any of claims 1 to 4 for the measurement of hemodynamic instability before, during and after dialysis by monitoring blood characteristics in the radial artery and in blood vessels of the forearm.

12. Method according to any preceding claim, wherein said light sources are light emitting diodes (LED).

13. A PPG probe comprising at least one near-infrared light source, in conjunction with at least one pair of blue-green light sources and a single photo-detector, wherein said blue-green light sources are positioned proximal to said photo-detector and said near- infrared light source(s) is/are positioned distal to said photo-detector.

14. Probe according to claim 13, wherein said light sources are light emitting diodes (LED).

15. Probe as claimed in claim 13 or 14, wherein the near-infrared light source is placed within a distance of 5 mm to 50 mm of the detector and wherein the blue-green light sources are placed at a distance of between 0 mm to 5 mm of the detector.