WO2002043561A9

WO2002043561A9 - Measuring haematocrit in blood vessels

Info

Publication number: WO2002043561A9
Application number: PCT/US2001/043097
Authority: WO
Inventors: Warren Groner
Original assignee: Cytometrics Llc
Priority date: 2000-11-15
Filing date: 2001-11-15
Publication date: 2003-09-04
Also published as: CA2428866A1; WO2002043561A2; JP2004516052A; CN1529565A; EP1335665A2; MXPA03004286A; AU2002241474A1; WO2002043561A3

Abstract

A method, system, and computer program product is provided for analyzing of a microcirculatory system to determine the quantity of various blood components, such as red blood cells. The images are analyzed to identify vessel structure, and determine the variance in measurable parameters, such as the vessel diameter or optical density. In an embodiment of the present invention, the coefficient of variation is determined for the diameter measurements along the vessel and a fractional volume value is calculated for the red blood cells. The fractional volume value is used to estimate the hematocrit (Hct). In another embodiment, the coefficient of variation is determined from a plurality of optical density measurements made at multiple points along the vessel, and the fractional volume value and Hct are calculated from the coefficient of variation of the optical density measurements. In yet another embodiment, the variation in optical density is measured at a single point for a time series of images of a vessel, and a fractional volume and hematocrit are calculated from the coefficient of variation.

Description

MEASURING HAEMATOCRIT IN BLOOD VESSELS

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to reflected light analysis. More particularly, the invention relates to the use of reflected spectral imaging to determine a quantity of visualizable components within a fluid flowing in a tubular system. Still more particularly, the invention relates to the use of reflected spectral imaging to determine a quantity of components within the blood of a mammalian, especially human, vascular system.

2. Related Art

Widely accepted medical school doctrine teaches that the complete blood count including the white blood cell differential (CBC+Diff) is one of the best tests to assess a patient's overall health. With it, a physician can detect or diagnose anemia, infection, blood loss, acute and chronic diseases, allergies, and other conditions. CBC+Diff analyses provide comprehensive information on constituents in blood, including the number of red blood cells, the hematocrit, the hemoglobin concentration, and indices that portray the size, shape, and oxygen- carrying characteristics of the entire red blood cell (RBC) population. The

CBC+Diff also includes the number and types of white blood cells and the number of platelets. The CBC+Diff is one of the most frequently requested diagnostic tests with about two billion done in the United States per year.

A conventional CBC+Diff test is done in an "invasive" manner in which a sample of venous blood is drawn from a patient through a needle, and submitted to a laboratory for analysis. For example, a phlebotomist (an individual specially trained in drawing blood) collects a sample of venous blood into a tube containing an anticoagulant to prevent the blood from clotting. The sample is then sent to a hematology laboratory to be processed, typically on automated, multiparameter analytical instruments, such as those manufactured by Beckman- Coulter Diagnostics of Miami, Florida. The CBC+Diff test results are returned to the requesting physician, typically on the next day.

Invasive techniques, such as for conventional CBC+Diff tests, pose particular problems for newborns because their circulatory system is not yet fully developed. Blood is typically drawn using a "heel stick" procedure wherein one or more punctures are made in the heel of the newborn, and blood is repeatedly squeezed out into a collecting tube. This procedure is traumatic even for an infant in good health. More importantly, this procedure poses the risk of having to do a blood transfusion because of the low total blood volume of the infant. The total blood volume of the newborn infant is 60-70 cc/kg body weight. Thus, the total blood volume of low birth weight infants (under 2500 grams) cared for in newborn intensive care units ranges from 45- 175 cc. Because of their low blood volume and delay in production of red blood cells after birth, blood sampling from preterm infants and other sick infants frequently necessitates transfusions for these infants. Blood bank use for transfusion of infants in neonatal intensive care units is second only to the usage for cardiothoracic surgery. In addition to newborns, invasive techniques are also particularly stressful for, and/or difficult to carry out on, children, elderly patients, burn patients, and patients in special care units.

A hierarchical relationship exists between the laboratory findings and those obtained at the physical examination. The demarcation between the physical findings of the patient and the laboratory findings are, in general, the result of technical limitations. For instance in the diagnosis of anemia, it is frequently necessary to quantify the hemoglobin concentration or the hematocrit in order to verify the observation of pallor. Pallor is the lack of the pink color of skin which frequently signals the absence or reduced concentration of the heavily red pigmented hemoglobin. However, there are some instances in which pallor may result from other causes, such as constriction of peripheral vessels, or being hidden by skin pigmentation. Because certain parts of the integument are less affected by these factors, clinicians have found that the pallor associated with anemia can more accurately be detected in the mucous membrane of the mouth, the conjunctivae, the lips, and the nail beds.

A device which is able to rapidly and non-invasively quantitatively diagnose anemia directly from an examination of one or more of the foregoing areas would eliminate the need to draw a venous blood sample to ascertain anemia. Such a device would also eliminate the delay in waiting for the laboratory results in the evaluation of the patient. Such a device also has the advantage of added patient comfort.

Soft tissue, such as mucosal membranes or unpigmented skin, does not absorb light in the visible spectra and regions in the near-infrared. In particular, soft tissue does not absorb light in the spectral region where hemoglobin absorbs light. This allows vascularization to be differentiated by spectral absorption from surrounding soft tissue background. However, the surface of soft tissue strongly reflects light and the soft tissue itself effectively scatters light after penetration of only 100-500 microns. Therefore, in vivo visualization of the circulation is generally impractical because of the complexities involved in either finding suitable areas and/or compensating for multiple scattering and for specular reflection from the surface. Studies on the visualization of cells in microcirculation consequently have been almost exclusively invasive, using a thin section (less than the distance for multiple scattering) of tissue containing the microcirculation, such as the mesentery, that can be observed by a microscope using light transmitted through the tissue section. Other studies have experimented with producing images of tissues from within the multiple scattering region by time gating (see, Yodh, A. and B. Chance, Physics Today, March, 1995, 34-40). However, the resolution of such images is limited because of the scattering of light, and the computations for the scattering factor are complex. Some imaging studies have been done in the reflected light of the microcirculation of the nail beds on patients with Raynauds, diabetes, and sickle cell disease. These studies were done to obtain experimental data regarding capillary density, capillary shape, and blood flow velocity, and were limited to gross physical measurements on capillaries. No spectral measurements, or individual cellular measurements, were made, and Doppler techniques were used to assess velocity. The non-invasive procedure employed in these studies could be applied to most patients, and in a comfortable manner.

One non-invasive device for in vivo analysis is disclosed in U.S. Patent No. 4,998,533 to Winkelman. The Winkelman device uses image analysis and reflectance spectrophotometry to measure individual cell parameters such as cell size and number. Measurements are taken only within small vessels, such as capillaries where individual cells can be visualized. Because the Winkelman device takes measurements only in capillaries, measurements made by the Winkelman device will not accurately reflect measurements for larger vessels.

This inaccuracy results from the constantly changing relationship of volume of cells to volume of blood in small capillaries resulting from the non-Newtonian viscosity characteristic of blood. Consequently, the Winkelman device is not capable of measuring the central or true hematocrit, or the total hemoglobin concentration.

The Winkelman device measures the number of white blood cells relative to the number of red blood cells by counting individual cells as they flow through a micro-capillary. The Winkelman device depends upon accumulating a statistically reliable number of white blood cells in order to estimate the concentration. However, blood flowing through a micro-capillary will contain approximately 1000 red blood cells for every white cell, making this an impractical method. The Winkelman device does not provide any means by which platelets can be visualized and counted. Further, the Winkelman device does not provide any means by which the capillary plasma can be visualized, or the constituents of the capillary plasma quantified. The Winkelman device also does not provide a means by which abnormal constituents of blood, such as tumor cells, can be detected.

Other non-invasive devices for in vivo analysis are disclosed in commonly assigned U.S. Patent No. 5,983,120, issued November 9, 1999 to Warren Groner and Richard G. Nadeau, and entitled "Method and Apparatus for Reflected

Imaging Analysis" (hereinafter referred to as "the '120 patent"), and in commonly assigned U.S. Patent Application No.09/401 ,859, filed September 22, 1999 in the names of Christopher Cook and Mark M. Meyers, and entitled "Method and Apparatus for Providing High Contrast Imaging" (hereinafter referred to as "the '859 application"). The disclosure of the '120 patent and the '859 application is incorporated herein by reference as though set forth in its entirety. The devices of the '120 patent and the '859 application provide for complete non-invasive in vivo analysis of a vascular system. These devices provide for high resolution visualization of blood cell components (red blood cells, white blood cells, and platelets), blood rheology, blood vessels, and vascularization throughout the vascular system. The devices of the ' 120 patent and the '859 application allow quantitative determinations to be made for blood cells, normal and abnormal contents of blood cells, as well as for normal and abnormal constituents of blood plasma. The devices of the '120 patent and the '859 application capture a raw reflected image of a blood sample, and normalize the image with respect to the background to form a corrected reflected image. An analysis image is segmented from the corrected reflected image to include a scene of interest for analysis. The method and apparatus disclosed in the '120 patent and the '859 application employ Beer's law to determine such characteristics as the hemoglobin concentration per unit volume of blood. The reflected images obtained with the devices of the '120 patent and the '859 application can also be useful in determining the number of white blood cells per unit volume of blood, a mean cell volume, the number of platelets per unit volume of blood, and the ratio of the cellular volume of blood to its total volume which is generally called the hematocrit.

Using Beer's law to quantitatively measure components of a blood vessel in a spectral image requires the components to be uniformly distributed throughout the vessel. For instance, Beer's law can be used to determine the hemoglobin concentration from in vivo measurements of optical density at an isobestic wavelength of the hemoglobin absorption spectrum. However, this technique presupposes the blood vessel is uniformly filled with red blood cells. Since the measurements are taken from a spectral image, it is paramount that this image contains a representative sample of blood components, i.e. red blood cells.

Should the blood vessel contain a non-uniform distribution of red blood cells, the spectral image would most likely not contain a representative sample of blood components. Moreover, the optical density measurements would fluctuate widely over time and individual measurements would not accurately reflect the subject's true hemoglobin concentration.

The size of the vessel diameter directly influences the distribution of blood components. In large vessels where the vessel diameter is many times the diameter of the blood cells, it has been shown that red blood cells, and hence the hemoglobin they contain, are uniformly distributed along and within the vessel. Therefore, a spectral image of a large blood vessel is prone to contain a representative sample of blood components, i.e. an average number of red blood cells. Beer's law, in this instance, can be used to produce an accurate measurement of hemoglobin concentration.

However, in smaller vessels, the variation in the number of red blood cells is more prominent. As the vessel diameter becomes smaller, a lesser number of red blood cells are able to pass side by side through the vessel. In the smallest vessels, only a single stream of red blood cells is able to pass. In smaller vessels, the number of red blood cells in a spectral image is likely to vary significantly over both time and length along the vessel. As a result, it is difficult to get a representative image of the subject's blood vessel. Therefore, the optical density and hemoglobin measurements from Beer's law are limited by imprecision.

In smaller vessels, the red blood cell count can be measured as an alternative to calculating the hemoglobin concentration. This can be accomplished by counting the number of red blood cells per unit length in a blood vessel. This technique is effective in the smallest vessels where only a single stream of red blood cells is able to pass.

In medium sized vessels, which have a vessel diameter considerably larger than the diameter of a single red blood cell yet are not large enough to be filled uniformly with red blood cells, neither the cell counting technique or the Beer's law method can be used without a substantial variation in results. However, it is this range of vessel diameters that are most easily accessible for visualization and measurement when one uses the methods of the '120 patent and the '859 application. Thus, there is a need in the art for a method and system for quantitatively analyzing select images of a fluid stream having a non-uniformly distributed concentration of cellular components by using non-invasive in vivo techniques.

SUMMARY OF THE INVENTION

The present invention is directed to analyzing reflected spectral images of a microcirculatory system to measure the volume and concentration of a blood vessel, including arteries, veins and capillaries. The method and system of the present invention quantitatively analyzes a fluid stream having a non-uniform distribution of cellular components. The present invention can be used to evaluate the cellular concentration in an unfilled blood vessel. Basically, the method and system of the present invention measures the vessel diameter and optical density at various locations along the axis of the vessel. The coefficient of variation in the diameter and/or optical density measurements are used to estimate blood characteristics, such as the hematocrit. The method is used to perform in vivo analyses of blood in vessels from a spectral image. The method of the present invention can also be used to perform in vitro analyses by imaging blood in, for example, a narrow tube or flow cell.

The method of the present invention can also be used to analyze other types of fluids containing visible suspended particles. The spectral imaging system can be used to analyze fluids for particulate impurities. It is only necessary that the walls of the fluid path be sufficiently transparent to permit light to pass through to image the fluid and any impurities flowing in the path.

Features and Advantages

A feature of the present invention is that it can be used to determine characteristics, such as the hematocrit through the use of reflected spectral imaging.

Another feature of the present invention is that it can be used to determine blood characteristics in vessels having a non-uniform distribution of blood components.

An advantage of the present invention is that it provides a means for the rapid, non-invasive measurement of clinically significant parameters of the CBC+Diff test. It advantageously provides immediate results. As such, it can be used for point-of-care testing and diagnosis. A further advantage of the present invention is that it eliminates the invasive technique of drawing blood. This eliminates the pain and difficulty of drawing blood from newborns, children, elderly patients, burn patients, and patients in special care units. The present invention is also advantageous in that it mitigates the risk of exposure to ADOS, hepatitis, and other blood-borne diseases.

A still further advantage of the present invention is that it provides for overall cost savings by eliminating sample transportation, handling, and disposal costs associated with conventional invasive techniques. A still further advantage of the present invention is that it provides for the measurement of the hematocrit without having to individually count the red bl ood cells. As such, the present invention can precisely determine the red blood cell count even if the individual red blood cells cannot be clearly imaged.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is described with reference to the accompanying drawings. In the drawings, like reference numbers indicate identical or functionally similar elements. Additionally, the left-most digit(s) of a reference number identifies the drawing in which the reference number first appears. FIG. 1 a shows a cross-sectional view of a medium blood vessel containing red blood cells;

FIG. lb shows a cross-sectional view of a small blood vessel containing red blood cells;

FIG. lc shows a cross-sectional view of a large blood vessel containing red blood cells;

FIG. 2 illustrates vessel diameter measurements being taken by an embodiment of the present invention;

FIG. 3 illustrates intensity measurements being taken by an embodiment of the present invention; FIG. 4 shows a flow chart representing the general operational flow for measuring the hematocrit from the variation in diameter measurements according to an embodiment of the present invention;

FIG. 5 shows a flow chart representing the general operational flow for . measuring the hematocrit from variations in optical density measurements according to an embodiment of the present invention;

FIG. 6 compares hematocrit measurements according to the present invention versus in vitro hematocrit measurements; and FIG. 7 shows a block diagram of an example computer system useful for implementing the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to a method and system for performing quantitative analyses, particularly non-invasive, in vivo analyses of a subject's vascular system. The in vivo measurements discussed herein can also be performed in vitro by imaging blood in, for example, a tube or flow cell, as would be apparent to a person skilled in the relevant art(s). The images can be obtained from a spectral imaging apparatus preferably, but not necessarily, of the type described in the '120 patent or the '859 application. Nonetheless, the image can be obtained from any type of imaging apparatus designed to produce a contrast image of a suspension of particles in a vascular system in either transmitted or reflected light. As disclosed in the '120 patent or the '859 application, the spectral imaging apparatus includes a light source that is used to illuminate the portion of the subject's vascular system to be imaged. The reflected light is captured by an image capturing means. Suitable image capturing means include, but are not limited to, a camera, a film medium, a photocell, a photodiode, or a charge coupled device camera. An image correcting and analyzing means, such as a computer, is coupled to the image capturing means for carrying out image correction, scene segmentation, and blood characteristic analysis.

The in vivo method of the present invention is carried out by imaging a portion of the subject's vascular system. For example, the image can be created from a sub-surface region of a subject's tissues or organs. The tissue covering the imaged portion is thus preferably transparent to light, and relatively thin, such as the mucosal membrane on the inside of the lip or the sclera of the eyeball of a human subject. As used herein, "light" refers generally to electromagnetic radiation of any wavelength, including the infrared, visible, and ultraviolet portions of the spectrum. A particularly preferred portion of the spectrum is that portion where there is relative transparency of tissue due to absences of water abortion, such as in visible and near-infrared wavelengths. It is to be understood that for the present invention, light can be coherent light or incoherent light, and illumination can be steady or in pulses of light. Human blood is made up of formed elements and plasma. There are three basic types of formed blood cell components: red blood cells (erythrocytes); white blood cells (leukocytes); and platelets. As noted above, red blood cells contain hemoglobin that carries oxygen from the lungs to the tissues of the body. White blood cells are of approximately the same size as red blood cells, but do not contain hemoglobin. A normal healthy individual will have approximately

5,000,000 red blood cells per cubic millimeter of blood, and approximately 7,500 white blood cells per cubic millimeter of blood. Therefore, a normal healthy individual will have approximately one white blood cell for every 670 red blood cells circulating in the vascular system. A complete blood count (CBC) without white blood cell differential reports eight parameters: (1) hemoglobin (Hb); (2) hematocrit (Hct); (3) red blood cell count (RBC); (4) mean cell volume (MCV); (5) mean cell hemoglobin (MCH); (6) mean cell hemoglobin concentration (MCHC); (7) white blood cell count (WBC); and (8) platelet count (Pit). The first six parameters are referred to herein as RBC parameters. Concentration measurements (measurements per unit volume of blood) are necessary for producing values for Hb, Hct, RBC, WBC, and Pit. Hb is the hemoglobin concentration per unit volume of blood. Hct is the volume of cells per unit volume of blood. Hct can be expressed as a percentage, i.e.,:

(red blood cell volume ÷ volume of blood) X 100% (Eqn. 1)

RBC is the number of red blood cells per unit volume of blood. WBC is the number of white blood cells per unit volume of blood. Pit is the number of platelets per unit volume of blood. Red blood cell indices (MC V, MCH, and MCHC) are cellular parameters that depict the volume, hemoglobin content, and hemoglobin concentration, respectively, of the average red blood cell. The red blood cell indices can be determined by making measurements on individual cells, and averaging the individual cell measurements. Red blood cells do not change volume or lose hemoglobin as they move through the vascular system. Therefore, red blood cell indices are constant throughout the circulation, and can be reliably measured in small vessels. The three red blood cell indices are related by the equation:

MCHC = MCH ÷ MCV (Eqn. 2)

Thus, only two red blood cell indices are independent variables.

To determine values for the six RBC parameters listed above, the following two criteria must be met. First, three of the parameters must be independently measured or determined. That is, three of the parameters must be measured or determined without reference to any other of the six parameters. Second, at least one of the three independently measured or determined parameters must be a concentration parameter (per unit volume of blood). Therefore, values for the six key parameters can be determined by making three independent measurements, at least one of which is a concentration measurement which cannot be made in a small vessel. As disclosed in the '120 patent or the '859 application, Hb and Hct can be directly measured by reflected spectral imaging of large vessels, while MCV and MCHC can be directly measured by reflected spectral imaging of small vessels. In this manner, three parameters are independently measured, and two of the parameters (Hb and Hct) are concentration parameters measured per unit volume of blood. As such, the six RBC parameters listed above can be determined in the following manner:

Hb Directly measured Hct Directly measured

RBC Hct ÷ MCV

MCV Directly measured

MCH MCV x (Hb÷Hct)

MCHC Hb ÷ Hct

Alternatively, as disclosed in the '120 patent or the '859 application, Hb can be directly measured by reflected spectral imaging of large vessels, and MCV and MCHC can be directly measured by reflected spectral imaging of small vessels. In this manner, three parameters are independently measured, and one of the parameters (Hb) is a concentration parameter measured per unit volume of blood. As such, the six RBC parameters listed above can be determined in the following manner:

Hb Directly measured

Hct Hb ÷ MCHC

RBC Hb ÷ (MCV x MCHC)

MCV Directly measured

MCH MCV x MCHC

MCHC Directly measured

Hemoglobin is the main component of red blood cells. Hemoglobin is a protein that serves as a vehicle for the transportation of oxygen and carbon dioxide throughout the vascular system. Hemoglobin absorbs light at particular absorbing wavelengths, such as 550 nm, and does not absorb light at other non- absorbing wavelengths, such as 650 nm. Under Beer's law, the negative logarithm of the measured transmitted light intensity is linearly related to concentration. As explained more fully in the ' 120 patent or the ' 859 application, a spectral imaging apparatus can be configured so that reflected light intensity follows Beer's law. Assuming Beer's law applies, the concentration of hemoglobin in a particular sample of blood is linearly related to the negative logarithm of reflected light absorbed by hemoglobin. The more 550 nm light absorbed by a blood sample, the lower the reflected light intensity at 550 nm, and the higher .the concentration of hemoglobin in that blood sample. The concentration of hemoglobin can be computed by taking the negative logarithm of the measured reflected light intensity at an absorbing wavelength such as 550 nm. Therefore, if the reflected light intensity from a particular sample of blood is measured, the concentration in the blood of such components as hemoglobin can be directly determined. The method and system of the present invention can be used to directly measure the hematocrit and can be used to quantitatively analyze a vascular system even if the measured components are not uniformly distributed. For example, the present invention can be used to measure the hematocrit of a blood vessel that does not have a uniform distribution of red blood cells. FIG. 1 a illustrates a typical segment of a blood vessel 100 of length L and diameter D. Vessel 100 has a discrete number (N) of red blood cells. The hematocrit (Hct) of vessel 100 can be expressed as NV_B ÷ (π/4)D²L, where V_B is the mean volume of a red blood cell. Hence, the hematocrit for any region of vessel 100 can be expressed by the following probability function:

F(N) = NV_B ÷ (π/4)D²L (Eqn. 3)

where N is a parameter that varies along the vessel length L at any given time, and also varies in time, at any given point along the vessel length L. The distribution of the variable N is best described by the poisson distribution wherein the variance is proportional to the square root of the variable. For instance, at any given time, a section of blood vessel 100 would have an average number of red blood cells measured by:

N = (Hct)(π/4)D²L ÷ V_B (Eqn. 4) The standard deviation of the mean N is proportional to the square root of N , and the coefficient of variation (C.V.) can be calculated as the standard deviation over the mean, or:

1

C. V. = -≡Ξr (Eqn. 5)

Combining the equations 4 and 5, the measure of the fluctuation or variation in the number of red blood cells can be shown by:

Thus, the coefficient of the variation of N is a function of the Hct and the vessel diameter. This variation will be manifested as a variation in the optical density or the diameter along an image formed of the vessel. Alternatively, it will manifest as a variation in either the diameter or the optical density in a time series of images of any one point in the vessel.

As discussed above, the use of reflected spectral imaging to measure Hb is based on the assumption that the blood vessels are uniformly filled. High variations in the number of red blood cells measured along the length of a vessel suggest that the vessel is not uniformly filled. Referring to equation 6, it can be seen that the coefficient of variation is inversely related to the vessel's diameter. Larger blood vessels, having diameters exceeding 50 microns, have low coefficient of variations which suggest they have a uniform distribution of Hb. FIG. lb illustrates a typical small blood vessel of diameter D. Small blood vessels are those having a diameter less than 6 microns. The dimensions of the vessels in this range are comparable to the dimensions of the red blood cells (shown as diameter d). Therefore, typically only a single stream of red blood cells is permitted to flow through the vessels. In this instance, one can use spectrophotometry (i.e., Beer's law) to calculate the MCHC but not to measure the Hct and Hb. Red blood cell concentration can only be determined by counting red blood cells along the vessel.

For medium sized vessels (such as vessel 100 shown in FIG. 1 a), between the range of 6 to 60 microns, the high variation (e.g., 10-20%) indicates a non- uniform distribution of Hb and complicates the use of Beer's law to estimate the

Hb or the MCHC. The vessel size is large enough to permit multiple streams of red blood cells which impair the use of spectrophotometry to determine precise measures of RBC by counting individual cells. In fact, vessels in this range can be as large as 2 to 15 red blood cells in diameter. For unfilled vessels, the method and system of the present invention can be used to estimate the Hct from the coefficient of variation and vessel diameter.

FIG. lc illustrates a typical large vessel of diameter D. In large vessels, discrete measurements of reflective properties, and hence spectrophotometry (i.e.,

Beer's law), can be used to estimate the Hb. For smaller blood vessels, the high variation in the number of red blood cells imposes a profound effect on estimating Hb as a function of the optical density measured from a reflected spectral image.

FIG. 2 illustrates a vessel's diameter being measured. As shown, "m" diameter measurements (202, 204, 206 and 208) are made along the axis of vessel 100.

FIG. 3 illustrates a vessel's intensity being measured. As shown, "m" intensity measurements are made along the axis of vessel 100. The original light

(302, 304, 306 and 308) is depicted as I,,,,, and the attenuated light (312, 314, 316 and 318) is depicted as I„. Referring to FIG.4, flowchart 400 represents the general operational flow of the present invention. More specifically, flowchart 400 shows an example of a control flow for measuring the hematocrit of a blood vessel.

FIG. 4 begins at step 401. At step 405, an image is retrieved from a memory source or image directory. The images can be obtained from an input file stored in a temporary or permanent memory location on a hard disk drive or removable storage device, such as a floppy diskette, magnetic tape, optical disks, or the like, as would be apparent to a person skilled in the relevant art(s). The input file also includes the subject number or other data used to identify the subject. Alternatively, the images can be obtained in real time from an imaging apparatus preferably, but not necessarily, of the type described in the ' 120 patent or the '859 application.

At step 410, multiple measurements are taken along the axis of the vessel to calculate the diameter at different segments. Referring back to FIG. 2, "m" diameter measurements (202, 204, 206 and 208) are made along the axis of vessel 100. As would be apparent to a person skilled in the relevant art(s), the number of measurements or segments should be sufficient to obtain an accurate measurement of the diameter. At step 415, the diameter measurements are analyzed to determine the coefficient of variation.

At step 420, the fractional volume of a cellular component (e.g., red blood cell) is determined for the vessel. Two methods can be used to determine the fractional volume. Under the first method, the fractional volume of the cellular component is determined at each segment by taking the reciprocal of the product of the coefficient of variation and the diameter measurements for each segment. Under the second method, the fractional volume of the cellular component can be determined by taking the reciprocal of the product of the coefficient of variation and the average of the diameter measurements.

At step 425, the Hct is calculated from the mean fractional volume. If the first method is used to determine the fractional volume, the average of all the fractional volume measurements is calculated and used to estimate the Hct. If, however, the second method is used, the mean fractional volume calculated at step 420 would be used to estimate the Hct. After the Hct is calculated, the control flow of flowchart 400 ends as indicated by step 495.

Referring to FIG. 5, flowchart 500 represents the general operational flow of another embodiment of the present invention. More specifically, flowchart 500 shows an example of a control flow for using optical density to measure the hematocrit of a blood vessel.

FIG. 5 begins at step 501. The control flow proceeds to step 405 through step 410 as discussed above with reference to FIG. 4. At step 510, the vessel is illuminated at multiple sections along its axis to measure the original and attenuated light intensities. Referring back to FIG.3, "m" intensity measurements are made along the axis of vessel 100. An intensity profile is created by computing the negative logarithm of the ratio of the measured attenuated light intensity and the original light intensity to produce the optical density. As would be apparent to a person skilled in the relevant art(s), the number of measurements or segments should be sufficient to obtain an accurate measurement of the optical density.

At step 515, the optical density measurements are analyzed to determine the coefficient of variation. At step 520, the fractional volume of the cellular component is determined at each segment by taking the reciprocal of the product of the coefficient of variation and the diameter measurements from each segment. As discussed in reference to FIG. 4, the fractional volume of the cellular component can also be determined at each segment by taking the reciprocal of the product of the coefficient of variation and the average of the diameter measurements.

At step 525, the average of all the fractional volume measurements is calculated to estimate the Hct. If, however, the second method is used to determine the fractional volume, the mean fractional volume determined at step 520 would be used to estimate the Hct. The control flow of flowchart 500 ends as indicated by step 595.

Hence, it can be demonstrated that the hematocrit of a subject can be determined from the coefficient of variation in optical density or the diameter, either along an image formed of a blood vessel, or in a time series of images of any one point in the vessel. FIG. 6 shows the results of a comparative study that implements the methods and systems of the present invention. During the study, in vitro measurements of blood samples were taken from nine subjects and used to determine their hematocrit. Using the methods of the present invention, in vivo images from the eye (i.e., sclera) of the nine subjects were obtained by orthogonal polarization spectroscopy, as described in the '120 patent or the '859 application. For each subject, five blood segments were selected to determine diameter and optical density measurements. The variation in the optical density along the respective blood vessel was also measured. Next, the reciprocal of the product of the variation and the diameter for each segment was computed. Then, the average of this quantity for each of the nine subjects was calculated to combine the results from the in vivo measurements into a single hematocrit estimate.

The results from the in vivo and in vitro measurements are shown in FIG. 6. The ordinate axis represents the hematocrit determined from the in vitro measurements, and the abscissa represents the hematocrit from the in vivo measurements. The slope for the hematocrit calculation is 0.91, which suggests that the in vivo measurements are close approximations for the in vitro measurements.

As is apparent from the foregoing description, the present invention was developed primarily to analyze blood components in a non-invasive manner. However, it will be clear to persons skilled in the relevant art(s) that the analysis techniques of this invention have utility beyond the medical applications described above. The invention has application outside the medical area and can be used generally to quantitatively analyze visualizable components in a fluid flowing in any vascular system, such as a tube, the walls of which are transparent to transmitted and reflected light. The present invention is most effective for analyzing vessels having diameters between 6 to 60 microns, which represents the most likely sized vessel to be detected with spectrophotometry. A preferred range is 15 to 50 microns where the coefficient of variation averages 10 to 20%.

The present invention can be implemented using hardware, software or a combination thereof and can be implemented in one or more computer systems or other processing systems. In fact, in an embodiment, the invention is directed toward one or more computer systems capable of carrying out the functionality described herein.

Referring to FIG. 7, an example computer system 700 useful in implementing the present invention is shown. Computer system 700 includes one or more processors, such as processor 704. The processor 704 is connected to a communication infrastructure 706 (e.g., a communications bus, cross-over bar, or network). Various software embodiments are described in terms of this exemplary computer system. After reading this description, it will become apparent to a person skilled in the relevant art(s) how to implement the invention using other computer systems and/or computer architectures.

Computer system 700 can include a display interface 702 that forwards graphics, text, and other data from the communication infrastructure 706 (or from a frame buffer not shown) for display on the display unit 730.

Computer system 700 also includes a main memory 708, preferably random access memory (RAM), and can also include a secondary memory 710.

The secondary memory 710 can include, for example, a hard disk drive 712 and/or a removable storage drive 714, representing a floppy disk drive, a magnetic tape drive, an optical disk drive, etc. The removable storage drive 714 reads from and/or writes to a removable storage unit 718 in a well-known manner. Removable storage unit 718, represents a floppy disk, magnetic tape, optical disk, etc. which is read by and written to removable storage drive 714. As will be appreciated, the removable storage unit 718 includes a computer usable storage medium having stored therein computer software and/or data.

In alternative embodiments, secondary memory 710 can include other similar means for allowing computer programs or other instructions to be loaded into computer system 700. Such means can include, for example, a removable storage unit 722 and an interface 720. Examples of such can include a program cartridge and cartridge interface (such as that found in video game devices), a removable memory chip (such as an EPROM, or PROM) and associated socket, and other removable storage units 722 and interfaces 720 which allow software and data to be transferred from the removable storage unit 722 to computer system 700.

Computer system 700 can also include a communications interface 724. Communications interface 724 allows software and data to be transferred between computer system 700 and external devices. Examples of communications interface 724 can include a modem, a network interface (such as an Ethernet card), a communications port, a PCMCIA slot and card, etc. Software and data transferred via communications interface 724 are in the form of signals 728 which can be electronic, electromagnetic, optical or other signals capable of being received by communications interface 724. These signals 728 are provided to communications interface 724 via a communications path (i.e., channel) 726. This channel 726 carries signals 728 and can be implemented using wire or cable, fiber optics, a phone line, a cellular phone link, an RF link and other communications channels. In this document, the terms "computer program medium" and "computer usable medium" are used to generally refer to media such as removable storage drive 714, a hard disk installed in hard disk drive 712, and signals 728. These computer program products are means for providing software to computer system 700. The invention is directed to such computer program products. Computer programs (also called computer control logic) are stored in main memory 708 and/or secondary memory 710. Computer programs can also be received via communications interface 724. Such computer programs, when executed, enable the computer system 700 to perform the features of the present invention as discussed herein. In particular, the computer programs, when executed, enable the processor 704 to perform the features of the present invention. Accordingly, such computer programs represent controllers of the computer system 700.

In an embodiment where the invention is implemented using software, the software can be stored in a computer program product and loaded into computer system 700 using removable storage drive 714, hard drive 712 or communications interface 724. The control logic (software), when executed by the processor 704, causes the processor 704 to perform the functions of the invention as described herein.

In another embodiment, the invention is implemented primarily in hardware using, for example, hardware components such as application specific integrated circuits (ASICs). Implementation of the hardware state machine so as to perform the functions described herein will be apparent to persons skilled in the relevant art(s).

In yet another embodiment, the invention is implemented using a combination of both hardware and software.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example, and not limitation. It will be apparent to persons skilled in the relevant art(s) that various changes in form and detail can be made therein without departing from the spirit and scope of the invention. Thus, the present invention should not be limited by any of the above described exemplary embodiments.

Claims

WHAT IS CLAIMED IS:

1. A method for optically analyzing at least one of a plurality of visualizable cellular components suspended in a fluid vascular system, the walls of which are substantially transparent to transmitted and reflected light, using an image capturing device that is capable of capturing images from the fluid vascular system, and a processing unit in communication with the image capturing device, comprising the steps of:

(a) receiving in the processing unit an image of the fluid vascular system captured by the image capturing device; (b) analyzing said image to identify at least one vessel;

(c) generating a plurality of diameter measurements from said vessel;

(d) computing a coefficient of variation from at least one of said plurality of diameter measurements and a plurality of optical density measurements generated from said vessel ;

(e) deriving a product from said coefficient of variation and said diameter measurements; and

(f) determining a fractional volume of one of a plurality of visualizable cellular components in the fluid vascular system from said product.

2. The method according to claim 1, wherein the fluid vascular system comprises blood flowing in a blood vessel of a mammalian vascular system, and the visualizable cellular components comprise blood components, including red blood cells, and wherein: step (a) comprises receiving in the processing unit an image of the blood components in a region of the fluid vascular system; step (e) comprises multiplying said coefficient of variation with each of said plurality of diameter measurements to calculate a series of fractional volume values for the blood components; and step (f) comprises determining a hematocrit estimate from said series of fractional volume values.

3. The method according to claim 1, wherein the fluid vascular system comprises blood flowing in a blood vessel of a mammalian vascular system, and the visualizable cellular components comprise blood components, including red blood cells, and wherein: step (a) comprises receiving in the processing unit an image of the blood components in a region of the fluid vascular system; step (e) comprises multiplying said coefficient of variation with an average of said plurality of diameter measurements to calculate said product; and step (f) comprises determining a hematocrit estimate from said fractional volume.

4. The method according to claim 1 , further comprising the step of: generating said plurality of diameter measurements or said plurality of optical density measurements along the length of said vessel.

5. The method according to claim 1 , further comprising the steps of: receiving a time series of images of said vessel; and generating said plurality of diameter measurements or said plurality of optical density measurements from a common point on said vessel in said time series of images.

6. The method according to claim 1 , wherein each of said plurality of diameter measurements ranges from 6 to 60 microns, preferably 15 to 50 microns.

7. For use with a light transmitting device for transmitting light through a fluid vascular system, and an image capturing device for capturing images from the fluid vascular system, a processing unit adapted for communication with the image capturing device for analyzing at least one of a plurality of visualizable cellular components in the fluid vascular system, the walls of which are substantially transparent to transmitted and reflected light, comprising: receiving means for receiving an image of the fluid vascular system captured by the image capturing device, analyzing means for analyzing said image to identify at least one vessel, generating means for generating a plurality of diameter measurements from said vessel, first computing means for computing a coefficient of variation from at least one of said plurality of diameter measurements and a plurality of optical density measurements from said vessel, multiplying means for deriving a product from said coefficient of variation and said plurality of diameter measurements, and second computing means for determining a fractional volume of one of a plurality of visualizable cellular components in the fluid vascular system from said product.

8. The apparatus according to claim 7, wherein the fluid vascular system comprises blood flowing in a blood vessel of a mammalian vascular system, and the visualizable cellular components comprise blood components, including red blood cells, and wherein: said receiving means comprises means for receiving in the processing unit an image of the blood components in a region of the fluid vascular system; said multiplying means comprises means for multiplying said coefficient of variation with each of said plurality of diameter measurements to calculate a series of fractional volume values for the blood components; and said second computing means comprises means for determining a hematocrit estimate from said series of fractional volume values.

9. The apparatus according to claim 7, further comprising means for generating said plurality of diameter measurements or said plurality of optical density measurements along the length of said vessel.

10. The apparatus according to claim 7, further comprising means for generating said plurality of diameter measurements or said plurality of optical density measurements from a point on said vessel, said point being common in a time series of images of said vessel.

11. A computer program product comprising a computer useable medium having computer readable program code means embedded in said medium for causing an application program to execute on a computer that analyzes at least one of a plurality of visualizable cellular components in a fluid vascular system, said computer readable program code means comprising: a first computer readable program code means for causing the computer to analyze an image of the fluid vascular system captured by an image capturing device to identify at least one vessel; a second computer readable program code means for causing the computer to generate a plurality of diameter measurements from said vessel; a third computer readable program code means for causing the computer to derive a coefficient of variation from at least one of said plurality of diameter measurements and a plurality of optical density measurements from said vessel; a fourth computer readable program code means for causing the computer to derive a product from said coefficient of variation and said plurality of diameter measurements; and a fifth computer readable program code means for causing the computer to determine a fractional volume of one of a plurality of visualizable cellular components in the fluid vascular system from said product.

12. A computer program product according to claim 11 , wherein the fluid vascular system comprises blood flowing in a blood vessel of a mammalian vascular system, and the visualizable cellular components comprise blood components, including red blood cells, and wherein: said fourth computer readable program code means comprises computer readable program code means for causing the computer to multiply said coefficient of variation with each of said plurality of diameter measurements to calculate a series of fractional volume values for the blood components; and said fifth computer readable program code means comprises computer readable program code means for causing the computer to determine a hematocrit estimate from said series of fractional volume values.

13. A computer program product according to claim 11, further comprising: a sixth computer readable program code means for causing the computer to generate said plurality of diameter measurements or said plurality of optical density measurements along the length of said vessel.

14. A computer program product according to claim 11, further comprising: a sixth computer readable program code means for causing the computer to generate said plurality of diameter measurements or said plurality of optical density measurements from a point on said vessel, said point being common in a time series of images of said vessel.