U.S. patent application number 14/634238 was filed with the patent office on 2015-11-26 for method and apparatus for determining markers of health by analysis of blood.
The applicant listed for this patent is ERYTHRON, LLC. Invention is credited to Howland D. T. JONES, Robert G. MESSERSCHMIDT.
Application Number | 20150338338 14/634238 |
Document ID | / |
Family ID | 54009795 |
Filed Date | 2015-11-26 |
United States Patent
Application |
20150338338 |
Kind Code |
A1 |
MESSERSCHMIDT; Robert G. ;
et al. |
November 26, 2015 |
Method and Apparatus for Determining Markers of Health by Analysis
of Blood
Abstract
Biomarkers of high blood pressure are measured to identify high
blood pressure of the subject based on one or more biomarkers. In
many embodiments, the response of the biomarker to blood pressure
occurs over the course of at least an hour, such that the high
blood pressure identification is based on a cumulative effect of
physiology of the subject over a period of time. The methods and
apparatus of identifying high blood pressure with biomarkers have
the advantage of providing improved treatment of the subject, as
the identified biomarker can be related to an effect of the high
blood pressure on the subject, such as a biomarker corresponding to
central blood pressure. The sample can be subjected to increases in
one or more of pressure or temperatures, and changes in the blood
sample measured over time.
Inventors: |
MESSERSCHMIDT; Robert G.;
(Los Altos, CA) ; JONES; Howland D. T.; (Rio
Rancho, NM) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ERYTHRON, LLC |
San Francisco |
CA |
US |
|
|
Family ID: |
54009795 |
Appl. No.: |
14/634238 |
Filed: |
February 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61946494 |
Feb 28, 2014 |
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61984244 |
Apr 25, 2014 |
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62005522 |
May 30, 2014 |
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62083720 |
Nov 24, 2014 |
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Current U.S.
Class: |
435/288.7 |
Current CPC
Class: |
G01N 2201/06113
20130101; A61B 5/0075 20130101; G01N 21/27 20130101; G01N 2201/068
20130101; A61B 5/4833 20130101; G01N 21/59 20130101; A61B 5/02
20130101; G01N 33/492 20130101 |
International
Class: |
G01N 21/27 20060101
G01N021/27; G01N 21/59 20060101 G01N021/59; G01N 33/49 20060101
G01N033/49 |
Claims
1. An apparatus to identify a biomarker from blood of a subject,
comprising: a processor comprising instructions to identify a
biomarker of a blood sample of the subject; an optically
transmissive material having a measurement surface to receive the
blood sample; one or more optical energy detectors coupled to the
processor and optically coupled to the measurement surface to
measure an amount of the biomarker on the measurement surface; one
or more light sources to generate one or more measurement light
beams; and one or more optical elements arranged to direct the one
or more measurement light beams from the one or more light sources
to the measurement surface and to direct light from the measurement
surface to the one or more optical energy detectors, wherein the
one or more optical elements is configured to measure a surface of
one or more red blood cells on the measurement surface with an
evanescent wave extending from the measurement surface to the one
or more red blood cells on the measurement surface and wherein the
biomarker comprises a biomarker of a membrane of a red blood
cell.
2. An apparatus as in claim 1 wherein the biomarker comprises a
plaque biomarker.
3. An apparatus as in claim 2 wherein the plaque biomarker
comprises a material of one or more of a foam-cell rich plaque, a
lipid-rich plaque, or a collagen-rich plaque.
4. An apparatus as in claim 1, wherein the biomarker comprises a
blood pressure biomarker.
5. An apparatus as in claim 4, wherein the processor comprises
instructions to identify the blood pressure biomarker in response
to an evanescent wave measurement of the blood sample on the
measurement surface.
6. An apparatus as in claim 4, wherein the processor comprises
instructions to identify the blood pressure biomarker in response
to a transmission measurement of the blood sample between the
measurement surface and an opposing surface.
7. An apparatus as in claim 4, wherein the processor is configured
to identify the biomarker in response to the blood sample
comprising one or more of red blood cells, serum or plasma, and
wherein the apparatus is configured to measure a first component of
the blood sample comprising an increased amount of red blood cells
in relation to the blood sample and a second component of the blood
sample comprising a decreased amount of red blood cells in relation
to the blood sample.
8. An apparatus as in claim 7, wherein the processor is configured
to measure the first component of the blood sample with one or more
of a first evanescent wave measurement or a first optical
transmission measurement and to measure the second component with
one or more of second evanescent wave measurement or a second
optical transmission measurement.
9. An apparatus as in claim 8, wherein the processor is configured
to measure the first component of the blood sample with the first
evanescent wave measurement and the first optical transmission
measurement and to measure the second component with the second
evanescent wave measurement and the second optical transmission
measurement.
10.-11. (canceled)
12. An apparatus as in claim 4, wherein the apparatus is configured
to measure the one or more red blood cells in the presence of an
anticoagulant in order to permit a plurality of red blood cells to
contact the measurement surface with each of said plurality of red
blood cells having an elongate surface extending along an elongate
dimension aligned with the measurement surface in order to measure
a membrane of said each of the plurality of red blood cells.
13. An apparatus as in claim 1, further comprising: a prism
comprising the optically transmissive material, the prism
comprising the measurement surface to receive the blood sample.
14. An apparatus as in claim 13 wherein the prism comprises a Dove
prism and wherein the Dove prism is illuminated at a first angle
with a first light beam to measure one or more red blood cells on
the measurement surface with the evanescent wave of light
internally reflected from the measurement surface and wherein the
Dove prism is illuminated with a second light beam at a second
angle to measure light transmitted through the measurement surface
and the blood sample.
15. An apparatus as in claim 14, wherein the Dove prism comprises
an elongate axis and a width transverse to the elongate axis and
wherein the axis and the measurement surface extend in a first
direction and the width extends in a second direction transverse to
the first direction.
16. An apparatus as in claim 15, wherein the Dove prism comprises a
first inclined surface extending at a first angle oblique to the
measurement surface, and a second inclined surface extending a
second angle oblique to the measurement surface and wherein the
light beam illuminates the surface with total internal reflection
to provide the evanescent wave and the light from the evanescent
wave is transmitted through the second inclined surface.
17. An apparatus as in claim 16, wherein the measurement surface is
located between the first inclined surface and the second inclined
surface and wherein an opposing surface extends between the first
inclined surface and the second inclined surface opposite the
measurement surface and wherein a transmission measurement light
beam is transmitted through the opposing surface and the
measurement surface to measure transmission through the sample.
18. An apparatus as in claim 4, the processor comprises
instructions pressurize the sample and to identify the blood
pressure biomarker based on one or more temporal denaturation
profiles in response to pressurization of a sample chamber and
wherein the optically transmissive material is dimensioned to
pressurize the blood sample.
19. An apparatus as in claim 4, wherein the one or more light
sources and the one or more detectors and the one or more optical
elements are configured to measure chemicals based on optical
spectroscopy that are correlated with presence of hypertension
and/or correlated with level of blood pressure.
20. An apparatus as in claim 4, wherein the processor comprises one
or more instructions to one or more of transform or calibrate
biomarker concentration data into a corresponding scale related a
systolic and a diastolic pressure during the cardiac cycle, such
that the biomarker concentration is transformed from a
concentration to a blood pressure in response to measured data of a
plurality of subjects.
21. An apparatus as in claim 4, wherein the blood pressure
biomarker comprises one or more of adenosine triphosphate, or, one
or more transmembrane proteins, one or more proteins of the
membrane skeleton, one or more lipids of the red blood cell
membrane, a relative ratio of the one or more lipids of the red
blood cell membrane, or biomaterial deposited on the surface of the
red blood cell membrane.
22. An apparatus as in claim 1 wherein the biomarker comprises a
spectral signal and wherein the instructions comprise instructions
to identify the biomarker in response to the spectral signal.
23. An apparatus as in claim 1 wherein the biomarker comprises a
plurality of biomarkers comprising a spectral signal and wherein
the instructions comprise instructions to identify the plurality of
biomarkers in response to the spectral signal.
24. An apparatus as in claim 1, wherein the biomarker comprises one
or more of a protein, a lipid, a high density lipoprotein, a low
density lipoprotein, membrane protein, a transmembrane protein, or
a spectrin network, and wherein spectra of the biomarker comprise
one or more of an Amide I peak, an Amide II peak, a Carboxylate
peak, or an Amide III band.
25. An apparatus to identify a biomarker from blood of a subject,
comprising: a processor comprising instructions to identify a
biomarker of a blood sample of the subject, wherein the biomarker
comprises one or more of a protein, a lipid, a high density
lipoprotein, a low density lipoprotein, membrane protein, a
transmembrane protein, or a spectrin network, and wherein spectra
of the biomarker comprise one or more of an Amide I peak, an Amide
II peak, a Carboxylate peak, or an Amide III band, and wherein the
Amide I peak, the Amide II peak, and the Amide III band correspond
to an alpha helix of the biomarker, a beta sheet of the biomarker,
and disordered protein of the biomarker, respectively.
26. An apparatus as in claim 25 wherein the instructions comprise
instructions to identify the biomarker in response to one or more
of the Amide I peak, the Amide II peak, the Carboxylate peak, or
the Amide III band.
27. An apparatus as in claim 1 wherein the biomarker comprises a
spectral signal and wherein the instructions comprise instructions
to determine a plurality of factors in response to the spectral
signal.
28.-32. (canceled)
33. An apparatus as in claim 27, wherein the plurality of factors
comprises a plurality of multivariate curve resolution factors or a
plurality of multi-component analysis factors.
34. An apparatus as in claim 27 wherein the instructions comprise
instructions to identify the biomarker in response to a
relationship among a plurality of factors determined in response to
the spectral signal.
35.-47. (canceled)
48. The apparatus of claim 1, further comprising a sample container
to receive the blood sample of the subject.
49. An apparatus as in claim 48, wherein the sample container
extends in a vertical direction a distance sufficient to
gravimetrically separate red blood cells from serum of the blood
sample.
50. An apparatus as in claim 49, wherein the sample container
extends in a vertical direction a distance sufficient to wash the
red blood cells when the red blood cells separate from the serum
and wherein the container comprises a volume of a solution
sufficient to wash the red blood cells.
51.-53. (canceled)
54. An apparatus as in claim 48, wherein the sample container
comprises a removable container comprising a waveguide to introduce
the evanescent wave into a lower portion of the removable
container, wherein the waveguide comprises the optically
transmissive material having the measurement surface.
55.-72. (canceled)
73. An apparatus as in claim 1, wherein the processor comprises
instructions to identify the biomarker in response to a time series
of spectra of the blood sample as water is removed from the blood
sample.
74. (canceled)
75. An apparatus as in claim 1, wherein the processor comprises
instructions to identify the biomarker with water at least
partially removed from the blood sample to inhibit a water signal
of the blood sample and to inhibit lysing of the one or more red
blood cells, the processor comprising instructions to measure the
blood sample with the red blood cell membranes substantially intact
and adsorbed on the measurement surface with the water at least
partially removed from the blood sample.
76.-168. (canceled)
Description
CROSS-REFERENCE
[0001] The present PCT application claims priority to U.S. App.
Ser. No. 61/946,494, filed on Feb. 28, 2014, entitled "METHOD OF
DETERMINING BLOOD PRESSURE AND OTHER MARKERS OF CARDIOVASCULAR
HEALTH BY CHEMICAL ANALYSIS OF BLOOD SERUM" (attorney docket no.
45006-704.101); U.S. App. Ser. No. 61/984,244, filed on Apr. 25,
2014, entitled "METHOD AND APPARATUS FOR DETERMINING HEALTH BY
ANALYSIS OF BLOOD" (attorney docket no. 45006-704.102); U.S. App.
Ser. No. 62/005,522, filed on May 30, 2014, entitled "METHOD AND
APPARATUS FOR DETERMINING HEALTH BY ANALYSIS OF BLOOD" (attorney
docket no. 45006-704.103); and U.S. App. Ser. No. 62/083,720 filed
on Nov. 24, 2014, entitled "METHOD AND APPARATUS FOR DETERMINING
MARKERS OF HEALTH BY ANALYSIS OF BLOOD" (attorney docket number
45006-704.104); the entire disclosures of which are incorporated
herein by reference.
[0002] The subject matter of the present application is related to
PCT Application PCT/US2014/047097, filed on Jul. 17, 2014, entitled
"SPECTROSCOPIC MEASUREMENTS WITH PARALLEL ARRAY DETECTOR" (attorney
docket number 45006-703.601), the entire disclosures of which are
incorporated herein by reference.
BACKGROUND
[0003] The field of the present invention is related to biomarkers
of health, and more specifically to one or more of detecting,
diagnosing, screening, tracking over time, or ruling out, one or
more conditions such as high blood pressure and the harmful
cardiovascular effects of high blood pressure. Examples of harmful
effects of high blood pressure can include one or more of
inflammation, coronary artery disease, stable plaques, unstable
plaques, or other vascular factors related to the onset of heart
disease and heart attack in humans.
[0004] Prior methods and apparatus of measuring biomarkers are less
than ideal in at least some respects. Prior methods and apparatus
of measuring blood pressure and diagnosing subjects can be less
than ideal in at least some instances. Although blood pressure
measurements can be used to assess the health of a subject and
guide treatment, the prior methods and apparatus can be less than
ideal. Work in relation to embodiments as described herein suggest
that the prior peripheral blood pressure measurements can be less
than ideally suited to guide therapy of a target tissue. For
example, some organs such as the heart receive blood from the
central vasculature and the prior peripheral blood pressure
measurements may be less than ideally suited to guide therapy to
such organs. Also, pressure measurements may be less than ideally
suited to guide at least some treatments having a physiological
effect on the subject's health, and measuring blood pressure is a
somewhat indirect way of measuring subject physiology and
characteristics that may be related to the tissues and blood of
subject.
[0005] Modern blood pressure measurements are based on the
sphygmomanometer, also referred to as a blood pressure cuff. The
sphygmomanometer was invented by Samuel Siegfried Karl Ritter von
Basch in 1881. A sphygmomanometer in the form of a cuff was
patented in 1955 (GB740181). Although the sphygmomanometer remains
a very important tool in medicine, it can have problems and
deficiencies in at least some instances.
[0006] The sphygmomanometer in combination with a stethoscope
allows a trained health professional to measure two characteristic
values related to blood dynamics, the systolic and the diastolic
pressure. The health care practitioner attaches the cuff around the
subject upper arm over the brachial artery. Practitioner pumps up
pressure in the cuff until the brachial artery is completely
occluded. While listening to the brachial artery at the inside
crease of the elbow, practitioner slowly releases pressure in the
cuff. As the pressure falls, a whooshing sound is heard. These
so-called Korotkoff sounds occur when blood flow first starts again
in the artery. The pressure at which this sound is first heard is
noted as the systolic blood pressure. The cuff pressure is released
further until the Korotkoff sounds can no longer be heard. This is
noted as the diastolic blood pressure. The peak pressure in the
arteries is the systolic pressure, and the lowest pressure (at the
resting phase of the cardiac cycle) is the diastolic pressure. The
systolic and diastolic pressure measurements have become the
medical standard of care for diagnosing high blood pressure.
[0007] Although helpful in diagnosing high blood pressure, the
systolic and diastolic blood pressure measurements can result in
less than ideal measurements that may be related to one or more of
the following:
[0008] Observer error;
[0009] Systematic intraobserver and interobserver errors;
[0010] Terminal digit preference, rounding to favorite digit;
[0011] Observer prejudice;
[0012] White coat hypertension--high only in doctor's office;
[0013] Masked hypertension--normal in office, high at other times
of day;
[0014] Instrument error;
[0015] Defective control valve;
[0016] Improper fit of cuff, too large or too small;
[0017] Inadequate length of tubing;
[0018] Connections not airtight;
[0019] Position of manometer causes reading error;
[0020] Placement of cuff error;
[0021] Diastolic dilemma--muffling of sounds can occur 10 mm before
complete disappearance;
[0022] Two arms can exhibit different readings; or
[0023] Deflation too rapid.
[0024] These errors can lead to inaccurate blood pressure readings
that may be related to improper diagnoses in at least some
instances. For example, errors as large as 20 mm Hg may occur in at
least some instances.
[0025] If a subject is incorrectly diagnosed as having high blood
pressure when actually having low blood pressure, this person may
be placed on a daily blood pressure medication. Many of these
medications may have side effects, and more people than would be
ideal can be subjected to the side effects of blood pressure
medications. Also, blood pressure measurement errors may result in
a person who actually has high blood pressure being misdiagnosed as
having low blood pressure. An incorrect diagnosis for a subject
with high blood pressure can result in that subject not receiving
appropriate medication, such that the high blood pressure may not
be untreated in at least some instances. Inappropriate management
of high blood pressure can result in injury to the subject and may
even be fatal in at least some instances, and it would be helpful
to have fewer misdiagnoses of high blood pressure.
[0026] Blood pressure measurements located at the brachial artery
may be less than ideally suited to guide treatment. For example,
the brachial artery is located away from the aorta other central
blood vessels and provides a less than ideal determination of
central blood pressure, and measuring systolic and diastolic
pressure in the brachial artery of the arm may be less than ideally
suited to diagnose central high blood pressure that can be related
to organ damage in at least some instances. Although beta blocker
medications can lower peripheral blood pressure and blood pressure
of the arteries in the arm, these medications may not lower central
blood pressure in at least some instances, and people treated with
beta blockers having normal brachial pressure may still experience
heart failure.
[0027] Work in relation to embodiments suggest that it would
desirable to have a record of blood pressure and of cardiovascular
health over a period of time, rather than an instantaneous
measurement like brachial cuff pressure.
[0028] Although blood chemistry is the gold standard for screening,
diagnosis, and therapy in health wellness and medicine, the prior
methods are less than ideal in at least some respects. Currently, a
blood panel is requested by a physician and the patient is
instructed to travel to a blood laboratory where a phlebotomist can
draw blood from the antecubital vein into a series of special
collection tubes. The blood is then sent to a central blood
chemistry laboratory where it is chemically analyzed using numerous
wet chemical assays that have been developed and validated over the
years. More recently, a small portion of these tests can be
performed in a physician's office using specialized machines
employing enzymatic assays. Such delivery of blood to various
locations can be less than ideal.
[0029] Blood chemistry testing is rapidly moving to the
point-of-care for many reasons. The biggest of these are cost and
compliance. Blood testing in the POC and eventually in the home
dives down healthcare costs, is trackable and reportable, is
immediate and actionable, sticky, and socially supportive compared
to central lab testing. But the problem that needs to be overcome
is that central lab methods generally do not translate to the POC
and the home, since they require much wet chemistry and expensive
instrumentation.
[0030] Measurement and detection of biomarkers can be done in
conjunction with modern computers and software. These prior
computers and software can less than ideally solve the technical
problem of the detection and identification of biomarkers related
health of a subject. The prior software and algorithms can be less
than ideally suited to determine the health of a subject in
response to data such as spectral data.
[0031] In light of the above, it would be desirable to provide
improved methods and apparatus for measuring biomarkers of a
patient, such as biomarkers useful in determining blood pressure.
Ideally such methods and apparatus would provide a more accurate
reading of blood pressure with less variability and fewer false
negatives and false positives for high blood pressure, provide a
more accurate determination of central blood pressure, allow
improved treatment and management of blood pressure, and provide an
indicator of blood pressure and cardiovascular health over
time.
SUMMARY
[0032] Embodiments are directed to measurement of samples in order
to determine one or more biomarkers related to health. In many
embodiments, the one or more biomarkers comprises a biomarker of a
cell membrane, such as a biomarker of a red blood cell membrane.
The biomarker may comprise one or more of a component of a cell
membrane, or a substance such as a molecule that interacts with the
membrane.
[0033] Embodiments can provide improved methods and apparatus of
identifying high blood pressure of a subject. In many embodiments,
one or more biomarkers of high blood pressure are measured in order
to identify high blood pressure of the subject. Identifying the
blood pressure of a subject based on one or more biomarkers has the
advantage of being more accurate and less susceptible to short term
fluctuations in physiology and user variability at the time of the
measurement. In many embodiments, the response of the biomarker to
blood pressure occurs over the course of at least an hour, for
example at least a day, such that the high blood pressure
identification is based on a cumulative effect of physiology of the
subject over a period of time such as an hour, a day or weeks, as
opposed to the very short amount of time during which a blood
pressure measurement is made at a clinic and can fluctuate. The
methods and apparatus of identifying high blood pressure with
biomarkers as disclosed herein have the advantage of providing
improved treatment of the subject, as the identified biomarker can
be related to an effect of the high blood pressure on the subject,
such as a biomarker corresponding to central blood pressure. The
sample can be subjected to increases in one or more of pressure or
temperatures, and changes in the blood sample measured over
time.
[0034] In many embodiments, the apparatus comprises a first
measurement channel to measure the blood sample near a measurement
surface with an evanescent wave of an internally reflected light
beam, and a second measurement channel to measure the blood sample
through a thickness of the sample with a transmission measurement.
The transmission measurement can be measured through the
measurement surface and the thickness of the sample, such that an
internally reflected measurement beam and a transmission
measurement beam overlap at least partially. In many embodiments,
the evanescent wave measurement comprises an evanescent wave
spectroscopy measurement and the transmission measurement comprises
a transmission spectroscopy measurement. While the measurement
surface and first channel and the second channel can be configured
in many ways, in many embodiments the measurement surface comprises
a measurement surface of a Dove prism and the internally reflected
measurement beam is transmitted through inclined surfaces on
opposing ends of the Dove prism.
[0035] In many embodiments, the blood sample comprises a first
component having red blood cells or clotted cells and a second
component comprising plasma or serum and each of the first
component and the second component is measured. Each of the
components can be measured with the evanescent wave spectroscopy
and the transmission spectroscopy in order to provide four
measurement channels.
[0036] In a first aspect, embodiments provide an apparatus to
identify high blood pressure of a subject. The apparatus comprises
a processor comprising instructions to identify a blood pressure
biomarker of a blood sample of the subject.
[0037] In another aspect, embodiments provide a method of
identifying high blood pressure of a subject. A blood pressure
biomarker of a blood sample of the subject is identified.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] A better understanding of the features and advantages of the
present disclosure will be obtained by reference to the following
detailed description that sets forth illustrative embodiments, in
which the principles of the disclosure are utilized, and the
accompanying drawings of which:
[0039] FIG. 1 shows a blood sample from a subject being placed on a
measurement surface in order to measure blood pressure biomarkers,
in accordance with embodiments;
[0040] FIG. 2 shows a side profile view and corresponding
dimensions of a red blood cell, in accordance with embodiments;
[0041] FIG. 3 shows measurement of a blood sample with a Dove prism
in order to identify high blood pressure biomarkers with a first
measurement channel and a second measurement channel, in accordance
with embodiments;
[0042] FIG. 4 shows red blood cells located on a measurement
surface to measure the red blood cells with an evanescent wave and
identify high blood pressure biomarkers of the red blood cell
membranes, in accordance with embodiments;
[0043] FIG. 5 shows an apparatus to measure blood pressure
biomarkers, in accordance with embodiments;
[0044] FIG. 6 shows a method of measuring blood pressure
biomarkers, in accordance with embodiments;
[0045] FIG. 7 shows a substantially circular cross-section through
a red blood cell, in accordance with embodiments;
[0046] FIG. 8 shows measurement of a red blood cell membrane and
related structures, in accordance with embodiments;
[0047] FIG. 9 shows an apparatus comprising a database and a user
interface to determine identify markers of red blood cells related
to health, in accordance with embodiments;
[0048] FIG. 10 shows light entering germanium (index of refraction
n=4) at an incident angle of 80 degrees, resulting in total
internal reflection and a very shallow 1/e penetration depth of the
resulting evanescent wave into the sample, in accordance with
embodiments;
[0049] FIG. 11A shows a sample gravimetric washing container and
spectrometer to measure a blood sample, in accordance with
embodiments;
[0050] FIG. 11B shows a container as in FIG. 11A removed from the
spectrometer;
[0051] FIG. 11C shows a draw tube, in accordance with
embodiments;
[0052] FIG. 11D shows sample delivery and cell washing, in
accordance with embodiments;
[0053] FIG. 12 shows a method of analyzing a sample, in accordance
with embodiments;
[0054] FIG. 13 shows a commercially available spectroscopy
apparatus suitable for combination, in accordance with
embodiments;
[0055] FIG. 14 shows example spectra of fat, milk, dried red blood
cells, red blood cells, red meat and red wine, in accordance with
embodiments;
[0056] FIG. 15 shows PCA analysis of blood samples with and without
aspirin, in accordance with embodiments;
[0057] FIG. 16A shows multivariate curve resolution (MCR) factors
of an aspirin study, in accordance with embodiments;
[0058] FIG. 16B shows MCR concentrations for the factors of FIG.
16A, in accordance with embodiments;
[0059] FIG. 17 shows a comparison between fresh and
gluteraldehyde-stiffened chicken red blood cells (measurement time
of one minute), in accordance with embodiments;
[0060] FIG. 18 shows the effect of aspirin on the red blood cell
membrane, in accordance with embodiments;
[0061] FIG. 19 shows shifts in factor 3, factor 6, and factor 10,
in accordance with embodiments;
[0062] FIG. 20 shows a 3D plot of spectral data normalized to the
Amide I peak for blood before and after gluteraldehyde addition, in
accordance with embodiments; and
[0063] FIG. 21 shows a 2D plot of the spectral data of FIG. 20;
[0064] FIG. 22 shows a method of spectral data analysis suitable
for incorporation with embodiments;
[0065] FIG. 23 shows results from a study of mean arterial blood
pressure measurements in human subjects using a sphygmomanometer or
blood pressure cuff;
[0066] FIG. 24 shows results from a study of mean arterial blood
pressure measurements in human subjects using a measurement
apparatus in accordance with embodiments; and
[0067] FIG. 25 shows additional results from the study of FIG.
24.
DETAILED DESCRIPTION
[0068] A better understanding of the features and advantages of the
present disclosure will be obtained by reference to the following
detailed description that sets forth illustrative embodiments, in
which the principles of embodiments of the present disclosure are
utilized, and the accompanying drawings.
[0069] Although the detailed description contains many specifics,
these should not be construed as limiting the scope of the
disclosure but merely as illustrating different examples and
aspects of the present disclosure. It should be appreciated that
the scope of the disclosure includes other embodiments not
discussed in detail above. Various other modifications, changes and
variations which will be apparent to those skilled in the art may
be made in the arrangement, operation and details of the method and
apparatus of the present disclosure provided herein without
departing from the spirit and scope of the invention as described
herein.
[0070] The embodiments disclosed herein can be combined in one or
more of many ways to provide improved measurements of blood samples
from a subject.
[0071] As used herein like characters identify like elements.
[0072] In many embodiments, an evanescent wave comprises a
near-field wave with an intensity having an exponential decay as a
function of the distance from the boundary at which the wave was
formed. Materials place on a surface can interact with the near
field wave, with or without absorption, for example. This use of
the evanescent near field wave can provide improved signal to noise
ratios when measuring the membrane of cells such as the red blood
cell. The localization of the evanescent wave intensity profile to
the cell membrane can provide an effective amplification of the
measured signal.
[0073] The shape of the red blood cell (hereinafter "RBC") is
particularly well suited for evanescent wave measurement as
disclosed herein. The red blood cell membrane comprises a biconcave
disk shape having a flattened region along the long dimension and
an indentation near center, which allows the red blood cells to
settle onto a measurement substrate such that the long dimension of
the red blood cell extends in a direction along the surface of the
substrate, such that a significant portion of the red blood cell
membrane along the long dimension can be exposed to the evanescent
wave and measured. The red blood cell membrane comprises proteins
and lipids, and this structure provides properties for
physiological cell function such as deformability and stability.
Approximately 2.4 million new erythrocytes are produced per second.
The cells develop in the bone marrow and circulate for about
100-120 days in the body before their components are recycled by
macrophages. The deformability of the human red blood cell results
from the dynamic interaction of the phospholipid bilayer plasma
membrane and the structural spectrin molecular network. Adenosine
5'-triphosphate (ATP) facilitates remodeling in the coupled lipid
and spectrin membranes.
[0074] As used herein, a red blood cell encompasses an
erythrocyte.
[0075] The embodiments disclosed herein can be combined in one or
more of many ways.
[0076] In many embodiments, the detection and diagnosis of disease
and wellness through reagent-less whole cell in vitro analysis of
changes in the erythrocyte membrane from a single drop of blood
collect via a lancing device is provided.
[0077] The embodiments as disclosed herein are particularly well
suited for performing spectroscopic analysis of RBC proteins,
lipids, and combinations thereof, for example for assessing the
risk of cardiovascular diseases. The spectroscopic analysis can be
performed without in vitro enzymatic analysis, and without lysing
the cells or pretreating samples, for example.
[0078] In many embodiments, spectroscopic analysis of the RBC for
detecting cell stress and changes in cell morphology associated
with hypertension allows retroactive assessment of past cell damage
due to elevated blood pressure. The retroactive assessment can
significantly decrease the need for continuous blood pressure
measurement, and in many embodiments can eliminate bias due to
patient's mood or emotional state. The membrane of the erythrocyte
undergoes molecular changes, or remodeling, in hypertension. These
changes appear to be a response to increased shear forces on the
cells as blood pressure increases. When erythrocytes undergo shear
stress in constricted vessels, they can release ATP, which causes
the vessel walls to relax and dilate so as to promote normal blood
flow.
[0079] In many embodiments, the RBC is used as messenger cell to
report disease markers which the RBC encounters during
circulation.
[0080] The apparatus embodiments as disclosed herein are
particularly well suited for performing analysis of red blood cells
as disclosed herein.
[0081] In many embodiments, the apparatus comprises a user
interface and one or more databases for performing one or more of
the analyses as disclosed herein.
[0082] In many embodiments, the red blood cells (erythrocytes) are
separated, for example with standard method, such as centrifuge.
Alternatively or in combination, whole blood is separated
gravimetrically such that the relatively heavier erythrocytes fall
onto the sampling surface as described herein.
[0083] While the analytical method and apparatus can be configured
and performed in many ways, in many embodiments, the methods and
apparatus are configured for one or more of measurement of
mechanical or molecular properties via infrared, near-infrared, UV,
Raman, Surface enhanced Raman, resonance Raman, fluorescence, NMR,
terahertz, far infrared, circular dichroism) or through a
mechanical test (mechanical stiffness), or through a thermal
property analysis (thermal gravimetric analysis TGA). In many
embodiments, the analytical methods and apparatus comprise
molecular spectroscopy methods and apparatus, such as one or more
of infrared, Raman or near-infrared spectroscopy, for example. The
methods and apparatus can be configured to perform one or more of
measurements in transmission, absorbance, photo acoustic, or
reflection mode, in internal reflection mode, for example.
[0084] In many embodiments, the erythrocyte membrane is measured
for changes. The erythrocyte membrane can undergo molecular changes
during one or more of many disease states. Examples of examples of
membrane changes related to disease states that can be measured in
accordance with embodiments include:
[0085] Average blood glucose (membrane protein glycosylation)
[0086] High blood pressure (membrane elasticity)
[0087] Inflammation (fibrinogen on surface of membrane)
[0088] Cerebrovascular disorders (fibrinogen binding on RBC
membrane)
[0089] Thrombosis (erythrocyte agglomeration)
[0090] Unstable plaque (lipid on surface of cell membrane)
[0091] Acetylsalicylic Acid (ASA) therapy (cell membrane .cndot.
slippery-ness .cndot.)
[0092] Malaria (cell deformation)
[0093] Dehydration (membrane water content)
[0094] Sepsis (erythrocyte sedimentation rate)
[0095] Blood bank aging
[0096] Myocardial infarction (rigidity)
[0097] Diabetes (rigidity)
[0098] Sickle cell anemia (deformation)
[0099] Malaria (deformation, lipid profile)
[0100] Exercise oxidative stress (loss of C.dbd.C bonds)
[0101] Antioxidant level (ceruloplasmin level)
[0102] Drug uptake (Codeine, chlorpromazine, imipramine,
mefloquine, and pyrimethamine, acetazolamide, methazolamide, and
chlorthalidone and the ocular pressure reducing agent,
dorzolamide)
[0103] Hemolytic Anemia (lipid ratios)
[0104] Preeclampsia (membrane rigidity)
[0105] Ionic balance (protein stricture)
[0106] pH (protein structure)
[0107] Alzheimers (AD) (levels of proteins in membrane
skeleton)
[0108] Malnutrition (kwashiorkor and marasmus) (elevated
Cholesterol/phospholipid ratio)
[0109] Hereditary Spherocytosis (deficiency of ankyrin, spectrin
and protein 4.2)
[0110] Hereditary Elliptocytosis (spectrin defects, glycophorin
deficiency)
[0111] Acanthocytosis (free cholesterol/phospholipid ratio)
[0112] Alcohol (association with lipid bilayer)
[0113] Coumadin therapy dosimetry
[0114] Whole blood viscosity
[0115] In many embodiments, the presence of undesirable effects of
high blood pressure on the vascular system can be identified in one
or more of many ways. In many embodiments, an amount of one or more
biomarkers of the blood can measured in order to identify high
blood pressure of the subject. For example, a level of biomarker in
the blood can provide an indication of high blood pressure, and in
many embodiments an amount of biomarker from a blood sample above a
threshold amount can identify the subject as having high blood
pressure. In many embodiments, the methods and apparatus to measure
the biomarker can provide an improved identification of blood
pressure with fewer false positives and false negatives than at
least some prior cuff measurements of the brachial artery, for
example.
[0116] Work in relation to embodiments as described herein suggests
that the red blood cells (hereinafter "RBCs") can be involved in
the signaling of high blood pressure, and the methods and apparatus
as described herein can measure one or more RBC markers related to
the RBC signaling of high blood pressure. For example, increased
mechanical pressure on the RBCs can induce the RBCs to release one
or more biomarkers such ATP, for example. The released ATP may
signal changes to the blood vessel walls, or transmit signals to
the blood vessel walls, or both, for example. Alternatively or in
combination, cell membranes of the RBCs may stiffen, thereby
indicating chemical changes in the cell membrane of the RBC.
Although these effects may not yet be fully understood, the RBC
signaling, reporting, and responding to high blood pressure can be
combined with measurements of the RBCs to identify high blood
pressure of the subject, in accordance with many embodiments as
described herein.
[0117] In many embodiments disclosed herein, a biomarker provides a
record of blood pressure and of cardiovascular health over a period
of time, rather than an instantaneous measurement like brachial
cuff pressure. In many embodiments, the metric or biomarker is
related to recent history of high blood pressure would be. For
example, a time period of 90-120 days can be particularly useful
for reasons similar to that Hemoglobin Alc marker is useful for
controlling blood sugar in diabetes. Such a marker can be
especially useful for providing health and lifestyle advice to a
patient. Such a marker can also be especially useful for ensuring
the proper dosage and efficacy of a drug used to treat high blood
pressure, and for determining compliance with taking a therapeutic
agent, in accordance with embodiments disclosed herein.
[0118] In many embodiments, RBCs are large and as they travel
through the vasculature, can come in contact with vessel walls that
leave chemical residue on the RBCs, for example. In this manner the
RBC membrane comprises markers to identify and determine the
chemistry of the lining of the vessels walls. In many embodiments,
when this transfer occurs, the RBCs comprise a marker of the
atherosclerotic plaque that can be used to report the presence of
atherosclerotic plaques within the blood vessels. The corresponding
chemical spectrum obtained from the RBCs can be used to
differentiate the presence of an unstable plaque from a stable
plaque, which spectra are chemically distinct, for example.
[0119] In many embodiments, atherosclerotic plaques comprise one or
more of three categories: foam-cell rich, lipid-rich, or
collagen-rich. In many embodiments, the distinct chemistry of each
plaque leaves distinct residue patterns on the outside of the red
blood cell membrane. Lipid-rich plaques have been associated with
dangerous unstable plaques. The residual material of the one or
more plaques can be deposited on the red blood cell membrane and
measured in accordance with embodiments described herein.
[0120] In some embodiments, a substance is injected into the blood.
The substance may comprise one or more of mild abrasive,
stickiness, or affinity to atherosclerotic plaques, for example.
The affinity can be specific to one or more of the plaques as
described herein. After a period of time, this substance can be
recovered from blood via a blood draw. By measuring the exterior of
these substance particles, the presence of unstable plaques can be
detected. The substance may comprise one or more of many known
substances such as one or more of many known sugars, for
example.
[0121] In many embodiments, the abrasive substance comprises a
non-toxic material that causes alteration to the blood to in order
to cause a heightened but temporary level of abrasion and
inflammation in the coronary arteries. In many embodiments, the
substance clears from the blood in a short time after the
measurement is made. An example of a suitable candidate substance
is a sugar, such as one or more of glucose, fructose, or mannose,
for example. High blood sugar can be a known condition in diabetes.
Although sugar is known to cause inflammation in the vasculature
and can increase agglomeration in red blood cells, sugar clears
naturally from the blood system, since it is metabolized
readily.
[0122] In many subjects, the lifetime of an RBC can be
approximately 90 to 120 days. The changes of the RBC due to high
blood pressure can be related to the relatively recent history of
high blood pressure over the course of the lifetime of the RBC. If
a medication is taken for high blood pressure, the characteristics
of the RBCs can revert to normal relatively quickly because of the
rapid turnover of these cells, for example. Alternatively or in
combination, an amount of the one or more signaling biomarkers
stored on or within the RBC, such as ATP, can be related to blood
pressure of the subject, for example related to shear stress of the
RBC during cardiac cycling of the RBC.
[0123] There may be changes in other blood constituents as well.
For example, stiffened RBCs can be somewhat abrasive in the
vessels, which can lead to inflammation and additional biomarkers
suitable for measurement in accordance with embodiments disclosed
herein. While many biomarkers can be measured in accordance with
embodiments disclosed herein, an example of such biomarker suitable
for measurement is C--reactive protein (hereinafter "CRP"), for
example.
[0124] Proteins in blood can also change conformation in response
to pressure. Proteins such as albumin which exists in high
concentration in blood may also be measured in order to identify
high blood pressure of a subject.
[0125] In many embodiments, one or more components of blood are
analyzed such as the serum component of blood, or the cellular
component of blood, or both, in order to determine the presence of
biomarkers of high blood pressure.
[0126] In many embodiments, and amount of blood such as a drop of
blood is provided for analysis. For example, an amount of blood can
be provided into a capillary tube which has been heparinized. The
RBCs can be caused to separate from the serum. An instrument
configured in accordance with embodiments as described herein can
pass a beam of light may through the capillary tube, to measure one
or more of the serum portion, or the cellular portion, or both, for
example. The capillary tube can be pressurized, and one or more of
the constituents in blood such as proteins may respond differently
to pressure when the blood has been subjected to high blood
pressure, such that a differential measurement obtained. For
example, a first measurement can be obtained at first pressure and
a second measurement obtained a second higher pressure higher than
the first pressure. For example, the first pressure can be
approximately atmospheric pressure, and the second pressure can be
greater than atmospheric pressure. In many embodiments, pressures
as high as 600 MPa can be used to cause the unraveling and
denaturation of proteins in the blood. The rates and dynamics of
these protein changes in response to applied external pressure can
be correlated with the blood having been subjected to high blood
pressure previously within the subject.
[0127] FIG. 1 shows a blood sample 30 from a subject being placed
on a measurement surface 100 in order to measure blood pressure
biomarkers. The blood sample is obtained from the subject. The
subject has a hand 10 from which a blood sample can be obtained,
for example. Although a hand is shown the blood sample can be
obtained in one or more of many known ways. The blood sample is
placed on a measurement surface.
[0128] In many embodiments, the measurement surface on which the
red blood cells 40 are placed comprises an optical prism 110 for
the purpose of channeling measurement light 115 under the blood,
through the prism, by internal reflection. Internal reflection
spectroscopy can make spectroscopic measurements at a shallow depth
beyond the prism surface, since an evanescent wave is set up at
that interface. This rapidly diminishing evanescent wave rapidly
diminishes with distance away from the prism surface. The resulting
spectrum is thereby resulting from only the material that is
resting closest to the prism. In our blood cell sample, the
spectrum would contain information mainly about the cell membrane
and not the cytoplasm. One proposed mechanism of action for
correlating with blood pressure is changes in the cell membrane of
the red blood cells as a biomarker. In many embodiments, the
membrane spectrum contains spectra of one or more biomarkers having
amounts corresponding to the blood pressure of the subject.
[0129] The measurement surface can be configured in one or more of
many ways to measure the sample. In many embodiments, the
measurement surface comprises a flat surface of an optically
transmissive material such as Silicon or Germanium, for example.
The optically transmissive material can be shaped in one or more of
many ways to provide the measurement surface as described herein.
For example, the optically transmissive material may comprise a
prism, a flat plate, a cube, a rectangle or a Dove prism, for
example.
[0130] In many embodiments, the sample is measured near the
measurement surface with total internal reflection spectroscopy
(hereinafter "TIR"). With TIR, the measurement light beam is
directed toward the surface at an angle so as to provide total
internal reflection of the light beam from the measurement surface.
Although the light beam is reflected internally from the surface,
the light beam can interact with the sample on the opposite side of
the surface from the light beam with an evanescent wave of the
light beam. The evanescent wave of the light beam extends beyond
the measurement surface by a distance related to the wavelength of
the measurement light beam. In many embodiments, the evanescent
wave extends beyond the surface so as to provide a penetration
depth of about 0.1.lamda. into the sample place on the measurement
surface, where .lamda. is the wavelength of light. The TIR light
may comprise one or more of visible light, near-infrared light,
mid-infrared light or far infrared light, for example. In many
embodiments, the light used comprises mid-infrared light having one
or more wavelengths within a range from about 2 .mu.m (micrometer)
to about 20 .mu.m, for example. The one or more wavelengths of
light may comprise a plurality of wavelengths of light to scan to a
plurality of depths of the sample.
[0131] With TIR spectroscopy, the depth of the measurement is
related to the measurement wavelength such that the membranes of
red blood cells on or near the surface can be measured. With a 2
.mu.m wavelength, the penetration depth is about 0.2 .mu.m such the
penetration depth of the TIR measurement does not extend beyond a
thickness of a red blood cell. With a 20 .mu.m wavelength, the
penetration depth is about 2 .mu.m such the penetration depth of
the TIR measurement corresponds to the approximate a thickness of a
red blood cell.
[0132] FIG. 2 shows a side profile view and corresponding
dimensions of a red blood cell 40. The red blood cell comprises an
approximately toroidal shape having a long dimension along an
elongate axis defining a length 42 of the red blood cell and a
short dimension along a transverse axis defining a thickness 44 of
the red blood cell. The length of the red blood cell is
approximately 7 (seven) microns and the width is approximately 2
(two) microns.
[0133] When the red blood cell is forced through an opening with
blood pressure such as an opening of a capillary channel sized
smaller than the red blood cell, the shape of the red blood cell
can change to allow the red blood cell to pass, and one or more
biomarkers such as ATP can be released. Alternatively or in
combination, high central blood pressure can result in one or more
of deformation of the red blood cell or surface changes to the red
blood cell related to the high central blood pressure of the
subject, and the biomarkers corresponding to these changes can be
measured in accordance with embodiments disclosed herein.
[0134] In many embodiments, the methods and apparatus are
configured to measure the surface of the red blood cells and
identify one or more components of the red blood cells
specifically. A sampling and measurement system can be configured
to first separate cells from serum or plasma through sedimentation,
then place a sample of blood cells onto one measuring stage and a
sample of serum onto another measuring stage, for example, so as to
provide separate measurements. The volume of blood sample can be
small, such as a drop that could be obtained by a lancet at a
finger. The stage holding the blood cells may comprise a horizontal
surface on which the blood cells can be placed as described herein.
The measuring stage holding the serum or plasma may comprise
another measuring surface for TIR or transmission measurements as
described herein, and combinations thereof, for example.
[0135] FIG. 3 shows measurement of a blood sample 30 with a Dove
prism 300 in order to identify high blood pressure biomarkers with
a first measurement channel and a second measurement channel. In
many embodiments, the first measurement channel comprises a TIR
measurement channel, and the second measurement channel comprises
an optical transmission channel extending through a thickness of
the sample. The Dove prism can provide a first inclined surface 305
and a second inclined surface 310 that allow the first measurement
light beam 315 to be totally internally reflected and directed to
the inclined surfaces at an angle that decreases reflection from
the inclined surfaces. The Dove prism, like many shapes, comprises
a surface 320 opposite the TIR measurement surface 100 that
receives a second measurement beam 325 for transmission through the
measurement surface and bulk of the sample. The Dove prism
comprises an elongate axis 330 extending axially through the
inclined surfaces and between the measurement surface and the
opposing surface.
[0136] In many embodiments, a transparent movable support 350 is
provided to shape an upper surface of the sample for transmission
of the second measurement light beam. The transparent movable
support may comprise a thickness suitable for pressurizing the
sample with a pressure surface 355 for measurements as described
herein. Alternatively, the transparent movable support can be thin
to shape the blood sample without pressurizing the blood sample,
for example a microscope slide.
[0137] Although a Dove prism is shown, the optical system can be
configured in one or more of many ways with one or more of prisms,
cubes, rhomboids or parallelepipeds, for example.
[0138] FIG. 4 shows red blood cells 40 located on a measurement
surface 100 to measure the red blood cells with an evanescent wave
generated from the total internal reflection of the measurement
light beam 115 in order to identify high blood pressure biomarkers
of the red blood cell membranes 46, in accordance with
embodiments.
[0139] The blood sample 30 can be prepared in one or more of many
ways for placement on the measurement surface. In some embodiments,
the measurement surface or a solution combined with the blood
sample comprises a clotting antagonist to inhibit blood clotting,
in order to allow measurement of red blood cells and to separate
the blood cells into a first component having a greater number of
red blood cells and a second component having a greater amount of
plasma as compared to the sample as drawn from the subject.
Alternatively, the blood sample can be allowed to clot such that
the sample comprises a first clot component and a second serum
component, in which the clotting factors of the plasma have been
substantially depleted to form the blood clot.
[0140] In many embodiments, the components of the serum 32 or
plasma 34 and the blood cells 40 are each measured. In many
embodiments, the plasma and blood cells can be separated at least
partially so as to provide different measurements for each, for
example separate simultaneous measurements of each.
[0141] In many embodiments, a second beam of light can be
transmitted through the blood sample. In these embodiments, a
spectrum representative of the bulk of the measurement cell is
obtained. The second stage can be a similar internal reflection
prism to measure the blood serum both by internal reflection and by
transmission. The transmission measurement represents the bulk of
the serum or plasma. In many embodiments, the proteins 36 in the
blood can begin to coat the prism as time progresses. Therefore the
internal reflection channel becomes a way of measuring the proteins
in blood with greater intensity than could be measured in the bulk
serum sample. Alternatively or in combination, the red blood cells
can sediment downward onto the measurement surface, and the
membranes of the red blood cells within the penetration depth of
the evanescent wave can be measured and the bulk of the plasma
measured with the transmission beam.
[0142] In many embodiments, two measurement cells on two
measurement stages can be used to measure the two components of
blood separately such that four measurements from four independent
measurement channels are provided. The evanescent wave measurements
can be combined with the transmission measurements so as to provide
four different spectral channels. Each of these channels can be
interrogated with different wavelengths of light, from the visible
to the far infrared region.
[0143] In many embodiments, each of these channels is measured as a
function of time to follow changes in the blood cells and the serum
and/or plasma with time. During this time, the samples can be
subjected to different temperatures by embedding a heating or
cooling element into the stages. Alternatively or in combination, a
movable transparent support 350 comprising an optical window can be
added on top of the blood cell and serum or plasma sample. This
support comprising the window can be mounted in a frame which can
create a pressure seal at the stage. In many embodiments, a high
external pressure can be exerted on the blood cells and blood
serum. Pressures of up to 600 MPa can be used in order to denature
and change the structure of the components and specifically
proteins in the sample, for example. In many embodiments, these
dynamic measurements can identify differences among biomarkers in
blood that has been exposed to high blood pressure versus blood
from subject without high blood pressure, for example.
[0144] FIG. 5 shows an apparatus 500 to measure blood pressure
biomarkers. The apparatus comprises a first measurement stage 505
comprising a surface 100 to receive a blood sample 30 of a subject.
In many embodiments, the apparatus comprises a second stage 510 to
receive a second sample of the subject as described herein. For
example, the first sample may comprise a red blood cell component
and the second sample may comprise a plasma component, in which the
red blood cell component comprises a greater amount of red blood
cells than the initial sample from the subject and the plasma
component comprises a greater amount of plasma than the initial
sample from the subject, for example. The first measurement stage
and the second measurement stage may comprise similar components
and can be coupled to light sources, optics and detectors similarly
and in accordance with embodiments as described herein.
[0145] The apparatus to identify blood pressure biomarkers
comprises one or more light sources, for example first light source
515 and second light source 520. The apparatus comprises one or
more input optics optically coupled to the light sources so as to
receive light from the light sources, for example first input
optics 525 for TIR measurements and second input optics 530 for
bulk transmission measurements. The apparatus comprises one or more
output optics optically coupled to the sample container to receive
the light from the sample, for example first output optics 535 to
receive the TIR light and second output optics 540 to receive the
transmission light. The one or more output optics are optically
coupled to one or more detectors, for example first detector 545
coupled to output optics 535 and second detector 550 coupled to
output optics 540.
[0146] The components of the apparatus 500 can be coupled to a
processor 555 comprising instructions to control the measurement of
the sample, for example of the first sample stage. In many
embodiments, the processor is configured and coupled to the one or
more light sources, the input optics, the output optics and the
detectors in order to measure optical spectroscopy of the sample.
The processor can be coupled to the first light source to control
the generation of light for TIR measurements. The processor can be
coupled to the second light source to control the generation of
light for the transmission measurements. The processor can be
coupled to the first input optics and first output optics to
control the input and output optics of the TIR measurements as
appropriate, for example when the input and output optics comprise
one or more movable or electro-optical components such as shutters,
gratings, etalons, mirrors, lenses, Bragg cells, prisms or
wavelength selective filters, for example. The processor can be
coupled to second input optics and second output optics to control
the input and output optics of the bulk transmission measurements
as appropriate, for example when the input and output optics
comprise one or more movable or electro-optical components such as
shutters, gratings, etalons, mirrors, lenses, Bragg cells, prisms
or wavelength selective filters, for example.
[0147] The processor can be coupled to the first detector to
measure the light from the TIR measurement and the second detector
to measure light from the bulk transmission measurement. The
detectors of the apparatus 500 such as the first detector 545 and
second detector 550 may comprise one or more of many known
detectors such as a one or more of photodiode, a phototransistor, a
charge coupled device (hereinafter "CCD") array, or conducting
metal oxide semiconductor arrays (hereinafter "CMOS" arrays), for
example. The detectors or the processor may comprise analog to
digital conversion circuitry to provide a digital measurement
signal to the processor.
[0148] The light sources of the apparatus 500 such as the first
light source 515 and second light source 520 may comprise one or
more of many known light sources such as lamps, diodes, lasers,
laser diodes, tunable lasers, optical parametric oscillators,
providing a suitable wavelength of light, for example in the mid
infrared as described herein. In many embodiments, one or more of
the light source or the input optics is coupled to the processor to
vary the wavelength of light, for example.
[0149] The apparatus 500 may comprise similar components connected
to the processor for the second measurement stage. Alternatively,
the first stage and the second can be interchangeable such that the
first measurement stage can be removed and replaced with the second
measurement stage.
[0150] The first measurement stage may comprise the prism 110,
sample container 400 and movable transparent support 350 as
described herein. The stage may comprise a coil 560 embedded in the
container to heat the sample 30 as described herein, and an
actuator 565 coupled to the movable transparent support to
pressurize the sample. A pressure sensor and a temperature sensor
can also be provided on the measurement stage to monitor the
pressure and the temperature of the sample. The prism may comprise
a Dove prism having the measurement surface 100 to provide the
evanescent wave and bulk transmission measurements as described
herein.
[0151] The processor comprises a tangible medium to store the
instructions, such as one or more of random access memory
(hereinafter "RAM"), read only memory (hereinafter "ROM"), flash
memory, gate array logic, a gate array, or a field programmable
gate array, for example. The processor may comprise a processor
system comprising a plurality of processor in communication with
each other, for example. In many embodiments the processors
communicate with each other with one or more known communication
methods and apparatus such as wireless communication, a shared bus,
a shared drive, serial communication, the Internet, and
combinations thereof, for example.
[0152] The changes in one or more components of blood disclosed
herein can be measured in one or more of many ways. For example,
the changes can be detected using a one or more of many types of
chemical analyses, such as spectroscopy and spectrometry, for
example. In many embodiments, spectroscopy methods and apparatus
are configured for measuring blood components, such as changes in
molecular conformation in blood cell membranes and blood proteins.
Examples of suitable spectroscopy methods and apparatus suitable
for incorporation in accordance with embodiments disclosed herein
include one or more of vibrational spectroscopy, either
mid-infrared or near-infrared absorption or reflection
spectroscopy, or Raman spectroscopy, and combinations thereof. In
many embodiments, vibrational spectroscopy methods and apparatus
are configured to measure levels of metabolites and proteins in
blood. In many embodiments, mass spectrometry methods and apparatus
are configured to measure one or more components of blood as
described herein. In many embodiments, nuclear magnetic resonance
(hereinafter "NMR") methods and apparatus can be configured to
determine the presence of biomarkers of the one or more components
of blood as described herein.
[0153] The spectroscopy may comprise one or more of molecular
spectroscopy (infrared, near-infrared, UV, Raman, Surface enhanced
Raman, resonance Raman, fluorescence, NMR, terahertz, far infrared,
circular dichroism). Additional or alternative testing can be used
such as a mechanical test (mechanical stiffness), or through a
thermal property analysis (thermal gravimetric analysis TGA), for
example, or rheology, for example.
[0154] FIG. 6 shows a method 600 of measuring biomarkers of blood
such as blood pressure biomarkers, in accordance with
embodiments.
[0155] At a step 605, a blood sample is provided. The blood sample
may comprise a single drop of blood.
[0156] At a step 610, the blood is separated into a first component
and a second component.
[0157] At a step 615, the sample is placed on the support.
[0158] At a step 620 a biomarker of the blood sample is
measured.
[0159] At a step 625, the first component is measured with one or
more of TIR or transmission spectroscopy.
[0160] At a step 630, the second component is measured with one or
more of TIR or transmission spectroscopy.
[0161] At a step 635, the sample is pressurized.
[0162] At a step 640, the sample is heated.
[0163] At a step 645, the sample profile is measured over time with
a plurality of measurements.
[0164] At a step 650, a first light beam is generated with a first
light source. The first light beam may comprise a TIR light beam as
described herein.
[0165] At a step 655, the first light beam is transmitted through
first input optics.
[0166] At a step 660, the sample is coupled with the first light
beam.
[0167] At a step 665, the first light beam is transmitted through
the first output optics.
[0168] At a step 670, the sample is measured with the first
detector.
[0169] At a step 675, a second light beam is generated with a
second light source. The second light beam may comprise a
transmission light beam for measuring a bulk thickness of the
sample as described herein.
[0170] At a step 680, the second light beam is transmitted through
second input optics.
[0171] At a step 685, the sample is coupled with the second light
beam.
[0172] At a step 690, the second light beam is transmitted through
the second output optics.
[0173] At a step 695, the sample is measured with the second
detector.
[0174] At a step 700, each of the components of the sample is
measured. For example each component can be measured with two
measurement channels as described herein.
[0175] At a step 705, the data are processed.
[0176] At a step 710, a blood biomarker such as a blood pressure
biomarker is identified. For example, the presence of the biomarker
can be determined in order to establish the presence or absence of
a biomarker.
[0177] At a step 715, the subject is treated.
[0178] The method 600 discloses a method of measuring blood
pressure in accordance with embodiments. A person of ordinary skill
in the art will recognize many variations and modifications based
on the disclosure provided herein. For example, some steps may be
added or removed. Some of the steps may comprise sub-steps, and
many of the steps can be repeated.
[0179] The processor as described herein can be programmed with one
or more instructions to perform one or more of the steps of the
method 600 of measuring blood pressure of the subject, for
example.
[0180] Therefore, the above steps are provided as an example of a
method of measuring blood pressure of the subject in accordance
with embodiments.
[0181] In many embodiments, a plurality of biomarkers is measured
to identify the presence of high blood pressure of the subject. For
example, a first biomarker can be measured and a second biomarker
can be measured. In many embodiments, an amount of the first
biomarker increases in response to the high blood pressure and an
amount of the second biomarker decreases in response to the high
blood pressure. Alternatively, amounts of both biomarkers can
increase, or both amounts can decrease, for example. In many
embodiments, a plurality of three or more biomarkers is measured,
and an amount of a first at least one biomarker increases above a
threshold amount to identify the high blood pressure and a second
amount of a second at least one biomarker decreases below a
threshold amount to identify the presence of the high blood
pressure.
[0182] The methods and apparatus as described herein can be
combined in one or more of many ways to measure one or more
biomarkers of high blood pressure, and the embodiments disclosed
herein provide examples, and a person of ordinary skill in the art
will recognize many modifications based on the disclosure provided
herein.
[0183] In many embodiments, one or more processors can be
configured with machine learning software in order to correlate
changes in the blood as exhibited in changes in the spectral
patterns, quantitatively with high blood pressure. This software
can use one or more the known tools of biostatistics, such as
principle components analysis (PCA), principle components
regression (PCR), partial least squares regression (PLS), classical
least squares (CLS), multivariate curve resolution (MCR), neural
networks, et cetera, for example.
[0184] In many embodiments, the biomarker for blood pressure
comprises a positive marker for blood pressure such that the
presence of the biomarker above a threshold amount indicates that
the subject has high blood pressure. Alternatively, the biomarker
for blood pressure comprises a negative biomarker for blood
pressure such the presence of the negative biomarker above a
threshold amount indicates that the subject does not have high
blood pressure. In many embodiments, a plurality of biomarkers are
measured in order to identify the presence (or absence) of high
blood pressure.
[0185] The positive or negative biomarkers, and combinations
thereof, can be identified in one or more of many ways as described
herein, such as with PCA, PCR, MCR, CLS, PLS or neural networks,
for example.
[0186] In many embodiments, the recent central aortic pressure
encompasses at least about one day of blood pressure, such that the
measure comprises an integral of subject blood pressure over at
least about one day based on a single blood draw. In many
embodiments, the recent central aortic pressure may comprise an
integral of blood pressure over a period of time of about 3 to 4
months. The recent blood pressure may comprise one or more of a
daily value or a 3-4 month period to determine long-term health and
wellness and property therapeutic value of drug interventions, and
durations in between for example. In many embodiments, the recent
blood pressure comprises at least about a 24 hour duration in order
to average out diurnal variations.
[0187] In many embodiments, the biomarker comprises one or more of
the following:
[0188] Adenosine diphosphate, one or more transmembrane proteins
(such as Band 3, Aquaporin 1, Glutl, ICAM-4, BCAM, Ankyrin, Band
4.1, Tropomyosin, Actin, or glycophorin), one or more proteins of
the membrane skeleton (such as spectrin), one or more lipids of the
red blood cell membrane, a relative ratio of the one or more lipids
of the red blood cell membrane, or biomaterial deposited on the
surface of the red blood cell membrane. Lipids in the RBC membrane
include Phosphatidylcholine (PC); Sphingomyelin (SM) in the outer
monolayer, and Phosphatidylethanolamine (PE), Phosphoinositol (PI)
(small amounts) and Phosphatidylserine (PS) in the inner membrane.
Approximately half the mass of the RBC membrane is proteins and
half is phopholipids. The ratio of protein to lipid may change with
high blood pressure, or the relative ratio of various lipids may
vary. For example the ratio of Phosphatidylcholine to Sphingomyelin
might be 60:40 in a healthy individual, but may change to 50:50 in
high blood pressure. Or the ratio of total lipid to total protein
may change from 50:50 in a healthy individual to 60:40 in high
blood pressure.
[0189] The biomarker may comprise one or more of specific changes
to the secondary structure of the transmembrane proteins, the
proteins of the membrane skeleton of the red blood cell, or changes
to the composition and relative ratios of membrane lipids of the
red blood cell membrane, and combinations thereof, for example.
Alternatively or in combination, the biomarker may comprise
biomaterial coated on the surface of the red blood cells that has
been deposited by contact with biomaterials inside the vasculature,
for example deposited in response to abrasive contact. In many
emodiments, the biomarker comprises one or more of a change to the
protein composition of the red blood cell membrane, a change to the
structure of the red blood cell membrane, a change to the structure
or composition of the lipids of the red blood cell membrane, an
endogenous biomaterial deposited onto the outside of the red blood
cell through contact during flow of the cells through the vessels,
or a foreign biomaterial deposited onto the outside of the red
blood cell through contact during flow of the cells through the
vessels, for example.
[0190] FIG. 7 shows a cross section of a red blood cell 40 in
accordance with embodiments. The circular cross section shows
structures of the red blood cell membrane 46, membrane proteins 50,
and structural proteins 54 within the red blood cell. The circular
cross sectional view shows the lipid bi-layer 48 of the red blood
cell membrane, which may comprise a phospholipid bi-layer for
example, cholesterol, and phosphatidyl choline, for example. The
ratio of components of the lipid bi-layer can be measured in
accordance with embodiments. The membrane protein 50 may comprise
one or more of many known membrane proteins, such as trans-membrane
proteins 52, for example. The membrane protein may comprise one or
more of Band 3, Ankyrin, CD47, Rh, or Glycophorin, for example. For
example, the red blood cell membrane may comprise trans-membrane
protein such as Ankyrin extending through the membrane in order to
transmit ions for example. The red blood cell membrane may comprise
interior protein such as spectrin protein, for example a spectrin
network 58 extending substantially along an interior of the cell
membrane and interior to the cell wall.
[0191] In many embodiments, the red blood cell membrane corresponds
to a fluid mosaic model of biological membranes, and membranes in
addition or alternative to the red blood cell membrane can be
measured. The membrane may comprise membrane proteins which are
mobile within the phospholipid and cholesterol layer. The spectrin
network of the membrane skeleton 56 provides strength to the red
blood cell membrane by interacting with the other proteins of the
membrane as described herein.
[0192] In accordance with embodiments, changes in the red blood
cell membrane and structures associated with the red blood cell
membrane can be measured. For example, lipids can be measured and
changes in lipids, lipid ratios and changes in lipid ratios,
proteins can be measured, protein ratios can be measured and
protein to lipid ratios can be measured.
[0193] The measurement in the analysis of the red blood cell
membrane can be performed in one or more many ways, for example,
with principal component analysis (PCA).
[0194] FIG. 8 shows an enlarged view of the red blood cell membrane
46 placed on a support structure 105 for measurement in accordance
with embodiments. The support comprises an optically transmissive
material as disclosed herein and the evanescent field 125, an
evanescent vector extending at least partially beyond an upper or
measurement surface 100 of the support on which the red blood cell
membrane reside. A light wave is infinite on the upper surface of
the support at an incidence angle 120 of theta. The measurement
light 115 comprises a wavelength lambda. The depth 135 of the
evanescent field comprises a zone of sensitivity 130. The zone of
sensitivity can be adjusted based on combinations of one or more of
the incidence angle .THETA. (theta) and the wavelength of light
(lambda), in order to limit the depth of the zone of sensitivity of
the measurement. The limitation of the measurement depth provides
measurement of the cell membrane on the surface, such as the red
blood cell membrane and corresponding structures such as the
trans-membrane proteins 52 and the structural proteins 54, and
inhibits measurement of deeper structures such as hemoglobin 60,
for example. The measured structures of the membrane can be
structures of the intact cell, and may comprise one or more of the
trans-membrane protein Ankyrin and the structural protein Spectrin,
for example.
[0195] The red blood cell may comprise an intact red blood cell as
described herein. The zone of sensitivity can inhibit measurement
of hemoglobin with a zone of sensitivity corresponding
substantially to the red blood cell membrane, the lipid bi-layer of
the red blood cell membrane, trans-membrane proteins of the red
blood cell membrane, and structural support proteins of the red
blood cell membranes, such as, spectrin for example. In many
embodiments hemoglobin is positioned within the intact red blood
cell at locations away from the red blood cell membrane such that
the zone of sensitivity does not extend substantially into a
hemoglobin molecule and, for example, does not extend across a
hemoglobin molecule within the red blood cell membrane. These
embodiments can provide specificity to the measurement and
localization to the red blood cell membrane.
[0196] In accordance with embodiments described herein, ratios of
components of the red blood cell or other membranes of another cell
can be measured. For example, the ratio of phosphatidyl choline to
cholesterol can be measured. The ratios of phospholipids to other
components can be measured such as the ratio of one or more lipid
components to a ratio of one or more protein components.
[0197] The components of the red blood cell membrane can be
measured in one or more of many ways, and reference is made to
spectroscopy merely by way of example in accordance with
embodiments.
[0198] Alternatively or in combination, rheology can be used to
measure the components of the red blood cell membrane. The rheology
measurement apparatus may comprise one or more capillary tubes
having a diameter size to inhibit flow and limit flow and provide
at least some resistance to blood flow, for example. The rheology
of the plurality of red blood cells measured may correspond to
structural aspects of the surface exterior, which can be affected
by one or more substances on the surface of the red blood cells,
for example.
[0199] The rheology components can be measured with a transform
function and transfer function. For example, the flow
characteristics of the red blood cells of the blood sample through
capillary tubes can be measured and the impedance profiles
determined for plurality of frequencies in order to determine a
transform function spectra. The impedance of the blood flow through
the one or more capillary tubes is measured at a plurality of
frequencies in order to provide a spectrum. The mechanical spectral
data can be combined with optical spectral data as described
herein. Alternatively, the mechanical spectral data can be used to
determine the presence of one or more biomarkers.
[0200] The rheology embodiments are well suited for combination
with the optical embodiments. For example, the aggregation of red
blood cells can affect the measured flow parameters of the blood,
and the aggregation of the red blood cells can also be related to
one or more surface components of the red blood cell membrane as
described herein, for example.
[0201] In many embodiments the analysis comprises a principal
component analysis (PCA), comprising the plurality of dimensions
and the dimensions may comprise orthogonal eigenvectors for
example. A person of ordinary skill in the art will have at least
some familiarity with PCA, and can determine the presence or
absence of biomarkers from a blood sample with PCA, for
example.
[0202] FIG. 9 shows an apparatus 900 comprising a database 905 and
a user interface 910 to determine identify markers of red blood
cells related to health in accordance with embodiments. The
apparatus 900 may optionally comprise one or more components of the
measurement apparatus 970 as disclosed herein, such apparatus 500,
for example. The user interface comprises a display 915 connected
to a processor 930 such that the user can view the biomarker data
920 on the display. The user interface also comprises one or more
user input fields 925. The processor may comprise a processor
system 935 and can store data of the database for the user to see
information of the database on the display. The processor comprises
a tangible medium 940 storing instructions of the database, such
that the user can see the information on the display. The tangible
medium may comprise a computer readable medium having one or more
of many known forms such as random access memory (RAM), read only
memory (ROM), compact disc CD-ROM, flash RAM. The processor may
comprise one or more of a plurality of Internet based cloud servers
945, a remote back end server 950, or a local server 955, or a
local processor 960 for example. The display may comprise a display
of a hand held processor such as a smart phone in communication
with a server, for example. Each of the components of the apparatus
900 can be connected in one or more of many ways as will be
apparent to a person of ordinary skill in the art, and each of the
components as shown can be connected to another component, either
directly or indirectly through other components and communication
pathways as disclosed herein.
[0203] The measurement apparatus as described herein can be
combined with the database and user interface in many ways. In many
embodiments, data from the measurement apparatus is shown on the
display. The data shown on the display may comprise data of the
amplified red blood cell measurement signal as described herein. In
many embodiments, output of the processor system, can be shown on
the display, in accordance with steps of one or more methods as
described herein, and the one or more processors may comprise
instructions to perform the one or more method steps and output the
data on the display. In many embodiments, the data output to the
user interface comprises cell membrane amplification data as
described herein, such as data of a plurality of cell membranes
shown on the display. The data of the plurality of cell membranes
may comprise evanescent wave data of a plurality of intact red
blood cell membranes, for example. In many embodiments, amplified
data comprises amplified cell membrane data of a plurality of
washed cells, such as gravimetrically separated washed red blood
cells as described herein. The data shown on the display to the
user may comprise one or more biomarkers of health from the
gravimetrically separated and washed membranes of intact red blood
cells, for example. The one or more processors as described herein
can be configured to with instructions stored on a tangible medium
such as a computer readable medium to provide the data on the
display.
[0204] FIG. 10 shows light 115 entering germanium optical structure
110 (index of refraction n=4) at an incident angle 145 of 80
degrees. This incident angle results in total internal reflection
and a very shallow 1/e penetration depth 135 of the resulting
evanescent wave 140 into the sample. The sample can comprise red
blood cells 40, as shown. The ends of the germanium can be
anti-reflection (AR) coated. The germanium optical structure may
comprise one or more inclined prism surfaces as described herein,
and may comprise waveguide as described herein, for example.
[0205] Table 1 shows penetration depths for various angles of
incidence and wavelengths in different sampler surfaces (diamond,
silicon, and germanium), in accordance with embodiments.
TABLE-US-00001 TABLE 1 Penetration Depths. Table 1. Penetration
Depths angle of depth of sample window sampler incidence
penetration index index wavelength surface (degrees) (microns) n2
n1 (microns) diamond 35 0.958 1.33 2.39 2 diamond 45 0.305 1.33
2.39 2 diamond 75 0.169 1.33 2.39 2 diamond 35 3.354 1.33 2.39 7
diamond 45 1.068 1.33 2.39 7 diamond 75 0.590 1.33 2.39 7 diamond
35 4.792 1.33 2.39 10 diamond 45 1.526 1.33 2.39 10 diamond 75
0.843 1.33 2.39 10 silicon 35 0.221 1.33 3.42 2 silicon 45 0.158
1.33 3.42 2 silicon 75 0.105 1.33 3.42 2 silicon 35 0.773 1.33 3.42
7 silicon 45 0.552 1.33 3.42 7 silicon 75 0.368 1.33 3.42 7
germanium 35 0.169 1.33 4.02 2 germanium 45 0.127 1.33 4.02 2
germanium 75 0.087 1.33 4.02 2 germanium 35 0.591 1.33 4.02 7
germanium 45 0.443 1.33 4.02 7 germanium 75 0.305 1.33 4.02 7
germanium 35 0.845 1.33 4.02 10 germanium 45 0.634 1.33 4.02 10
germanium 75 0.436 1.33 4.02 10
[0206] FIG. 11A shows a sample gravimetric washing container or
holder 400 and spectrometer 200 to measure a blood sample 30. In
many embodiments, the container is coupled to the spectroscopic
measurement apparatus as disclosed herein. The internally
reflective structure may comprise a waveguide 250 optically coupled
to the cells such as red blood cells 40 placed in the container.
The container comprises a vertically extending length 405 to
provide gravimetric separation. A cover or lid 410 extends over an
upper portion of the container. The cover comprises an opening 415
formed in the cover. A capillary tube may extend to the opening in
the cover.
[0207] In many embodiments the measurement apparatus comprises a
support fixed in relation to the spectrometer optics such that the
container can be removed. The support may comprise a lower support
425 fixed in relation to the optics of the spectrometer such that
the container can be placed on the lower support. The container may
comprise an upper support 420 affixed to the container such that
the container can be removed. The fixed lower support can be sized
to receive a portion of the container in order to engage the upper
support. The measurement apparatus comprises input coupling optics
230 such as a lens to couple the light source 210 of the
spectrometer to the waveguide structure of the container, and
output coupling optics 240 such as lens to couple the output of the
waveguide structure to photodetectors 220.
[0208] In many embodiments, the upper support, the lower support
and the coupling optics are arranged to couple the waveguide to the
coupling optics when the upper support rests on the lower support.
In many embodiments, the upper support comprises a lower flange or
rim of the container sized and shaped to be received with the lower
support and align the waveguide structure with the coupling optics
when received in the lower support.
[0209] Gravimetric separation can be performed in a solution 430.
The solution can be isotonic compared to blood, or can be
hypertonic or hypotonic compared to blood, and combinations
thereof. Hypertonic or hypotonic solution can result in
conformational changes in red blood cells which may be useful for
subsequent analysis. The solution can comprise saline. The solution
can comprise components with known spectral bands for spectroscopic
calibration, such as for example ethanol or methanol, and each
spectra can be determined in response to the known spectral bands,
for example. A container, of solution can be positioned on top of a
prism or other spectrometer sampling element, for example as shown
in FIG. 11A. The container can be shaped in one or more of many
ways and may comprise a cylindrical column, for example. The
container comprises a vertically extending length sufficient to
allow gravimetric separation of the red blood cells from other
components of the red blood cell sample such as the serum.
[0210] In many embodiments, the container column is placed on top
of a waveguide structure such as prism, for example. The container
may comprise a lower membrane having a thickness less than the 1/e
depth of the evanescent wave in order to measure the blood sample
through the membrane A thin optically transmissive material can be
located on the upper surface of the waveguide, in which the thin
material comprises a thickness less than the 1/e penetration depth
of the evanescent wave, for example.
[0211] The waveguide structure can be dimensioned in one or more of
many ways as disclosed herein. In many embodiments the waveguide
comprises a first end 252 to receive light energy and a second end
254 to transmit light energy. The wave guide may comprise an upper
surface 256 on an upper side oriented toward the sample and a lower
surface 258 on a lower side oriented away from the sample. The
waveguide may comprise a thickness extending between the upper
surface and the lower surface. In many embodiments the waveguide
comprises a length extending in a direction of propagation from the
first end to the second end. The waveguide may comprise a width
transverse to the length. In many embodiments, the waveguide
comprises a width greater than the thickness and a length greater
than the width in order to provide a plurality of internal
reflections of the measurement light energy from the upper surface
of the waveguide in order to amplify the optical signal transmitted
from the second end of the waveguide.
[0212] The ends of the waveguide can be configured in one or more
of many ways and may comprise surfaces extending perpendicular to a
long dimension of the waveguide, or inclined at an angle so as to
comprise prismatic surfaces. In many embodiments, the waveguide
comprises a prism, for example a dove prism as described
herein.
[0213] Alternatively or in combination, the removable container 400
may comprise the waveguide structure 250. The waveguide structure
can be removable with the container and located on the lower end of
the container. The container can be removed or placed with the
upper lid with comprising an upper hole or capillary for
introducing sample into the container. A sample comprising red
blood cells can be introduced to the container, and the relatively
heavier red blood cells can be separated gravimetrically and settle
onto the sampling surface either before or after the container has
been placed on the support.
[0214] In many embodiments, the red blood cells can be washed by
the solution during the gravimetric separation, such that potential
contaminants can be removed from the measurement.
[0215] FIG. 11B shows a container 400 as in FIG. 11A removed from
the spectrometer. In many embodiments, the container comprises a
removable container, such that the container comprises a single use
consumable item and the spectrometer components can be reused. In
many embodiments, the apparatus comprises a fixed support structure
that engages a removable support 420 affixed to the container. The
container can be accurately coupled to the spectrometer with a
support structure such as a flange, collar, or other support on the
container itself. The spectrometer and associated light source and
detector can be used to take measurements with the waveguide 250 on
the lower end of the container.
[0216] In many embodiments the lower support is fixed in relation
to the optics of the spectrometer, such that placement of the
container comprising the waveguide can be aligned with the
measurement optics when placed in order to provide accurate
spectroscopic measurements. One or more of the upper support or the
lower support can be sized and shaped in order to position the
waveguide with a position and orientation for measurement of the
cells on the lower surface of the container, for example.
[0217] Additional components can also be added to the container to
alter the sample if helpful. For example, gluteraldehyde can be
added to the column to alter red blood cell membrane structure.
[0218] In many embodiments, a plurality of gravimetric separation
containers is provided, in which each container of the plurality
comprises a removable single use consumable container.
[0219] In many embodiments, spectra can be measured from the sample
and statistical analysis methods can be used to generate a
plurality of factors. The plurality of factors may comprise a
plurality of functions upon which the data can be projected in
order to determine the amount, or concentration, of each function
in the sample. The factors can be orthogonal or non-orthogonal, for
example. The analysis can comprise one or more of principle
components analysis (PCA), principle components regression (PCR),
classical least squares (CLS), multivariate curve resolution (MCR),
partial least squares regression (PLS), neural networks, or other
biostatistical or chemometric approaches, for example. In many
embodiments, the factors are orthogonal to each other.
Alternatively, at least some of the factors may comprise
non-orthogonal factors. One or more relevant factors can be
identified, and the red blood cell status or history can be
determined in response to the one or more relevant factors. In many
embodiments, the history of the red blood cells comprises a control
of the red blood cells of the subject, for example a control of a
condition such as high blood pressure of the subject. The one or
more relevant factors may comprise one or more statistically
relevant factors, for example.
[0220] In many embodiments, a plurality of spectral bands comprise
peaks related to structure of the cell such as protein structure of
the red blood cell. The Amide I band of frequencies comprising the
Amide I peak may correspond to alpha helix protein structures of
the proteins of the red blood cell membrane. The Amide II band of
frequencies comprising the Amide II peak may correspond to
beta-sheet protein structures of the cell membrane. The band of
frequencies comprising the Amide III band may correspond to
disordered protein structures of the cell membrane. The
determination of factors corresponding to these spectral bands and
the shifts of peaks and intensities of these spectral bands in
response to the measure spectra can be used to determine the one or
more biomarkers of the cellular membrane such as the red blood cell
membrane.
[0221] In many embodiments, deformation of the red blood cell
membrane results in measurable spectroscopic changes to the red
blood cell membrane that can be measured as described herein. The
measurable changes may comprise shifts in the spectral peaks as
disclosed herein. The spectroscopic changes to the red blood cell
membrane can be substantially instantaneous, for example upon
deformation of the red blood cell membrane. Alternatively, the
spectroscopic changes to the red blood cell membrane may comprise
changes occurring over the history of the red blood cell, for
example over a long term three month history corresponding to the
90 to 120 day functional lifetime of the red blood cell.
[0222] In many embodiments the factors can be used to determine the
history of the red blood cell, and can be used to determine the
long term control of a condition such as hypertension, for example.
The long term control may comprise a conformational change to the
red blood cell membrane that can be determined with at least one
factor as disclosed herein, for example with a relationship among
factors as disclosed herein.
[0223] In many embodiments, the biomarker amplifies an optical
spectral signal. The biomarker may comprise a change to cell
membrane, such as a conformational change to a protein of a red
blood cell membrane or a ratio of components of the red blood cell
membrane as disclosed herein, for example. As the red blood cells
comprise a long dimension that can extend along the measurement
surface and optically couple the red blood cell membrane to the
evanescent wave measurement surface, the measured signal can be
amplified substantially. In many embodiments, a substance related
to the health status of the subject may not itself be detectable
with the spectral measurements. The measurement of the red blood
cell membrane can provide, however, an optical spectral signal to
determine the presence of the substance. For example, spectral
changes of the red blood cell membrane provided with aspirin as
disclosed herein can be used to identify a response of the red
blood cell membrane to aspirin, even though the presence of aspirin
itself may not be detectable spectroscopically in some embodiments.
The optical waveguide can be configured to provide a plurality of
reflections from the evanescent wave measurement surface in order
to provide an increased amplification of the measured evanescent
wave signal.
[0224] FIG. 11C shows a tube 440 to draw a sample. The draw tube
can be used to draw a blood sample 30, such as a sample from a pool
of blood on an external surface such as an external surface of a
finger 20. In many embodiments, the draw tube comprises a permeable
membrane having pores sized to wash the sample. Alternatively, the
draw tube may comprise an impermeable membrane for placement of the
sample in a container as described herein.
[0225] FIG. 11D shows sample delivery and cell washing with a
removable sample holder 400 as described herein. The sample holder
400 may comprise a container 450 coupled to an inlet tube 470 and
an outlet tube 475. The inlet tube can provide a rinse solution 480
and the outlet tube can pass rinsate 485 from the sample container.
The sample container may comprise an inner portion 455 and an outer
portion 460 with the permeable membrane 465 extending therebetween,
in order to provide cross-flow filtration, for example. The inlet
tube can be connected to the inner portion of the sample container
and the outlet tube can be connected to the outer portion of the
sample container. An attenuated total reflection (ATR) waveguide
crystal 250 can be located on a lower end of the sample container.
The cells of the sample 30 can be retained in the draw tube and
deposited onto the ATR crystal for measurement as described herein.
The rinsate column has the advantage of removing non-cellular
material from the measured sample, such as serum and potential
lysate.
[0226] The sample draw tube 440 as in FIG. 11C comprising the
semipermeable membrane 465 can be used to collect a blood sample
30, and the draw tube comprising the permeable membrane can be
placed in an annular container 450 comprising a column of fluid.
Alternatively, a drop of blood can be placed on an upper end of the
draw tube in order to receive the blood sample with the tube. The
permeable membrane may comprise an approximate pore size of about 5
um in order to inhibit passage of cells through the pores and to
allow passage of water and molecules, for example, in order to wash
the sample.
[0227] A cover 490 can be placed over the annular container in
order to wash the sample. The cover may comprise an inlet tube
extending from the cover. The cover may comprise an opening formed
therein coupled to a lumen 445 of the tube 440 placed into the
container 450, to pass fluid from the tube through the cover and
into the draw tube. An outlet can be coupled to an outer annular
portion of the annular container defined by the draw tube. The draw
tube can be placed within the annular container such that the lumen
of the draw tube defines a first inner portion of the annular
container within the draw tube and a second outer annular portion
of the annular container outside the draw tube.
[0228] The outlet tube can be connected to a lower portion of the
outer portion of the container as shown. Alternatively, the outlet
tube can be coupled to an upper portion of the sample container,
and may be integrated with the cover, for example, such that both
the inlet tube and the outlet tube extend from the cover.
[0229] The ATR waveguide crystal as described herein can be located
on a lower end of the annular container, and coupled to
spectrometer optics, such that the sample container comprises a
removable sample container among a plurality of sample containers
as described herein. The waveguide can be located on a lower end of
the draw tube, for example.
[0230] The sample holder 400 comprising the container has the
following advantages:
[0231] Washes the serum and potential lysate from the cell
membranes
[0232] Packs cells onto ATR crystal
[0233] Disposable
[0234] The sample container can be used with one or more of the
following steps:
[0235] Wash Cycle [0236] Washes serum and potential lysed material
into rinsate column;
[0237] Drain Cycle [0238] Drains a the rinsate column and in
addition drains a majority of the membrane straw leaving a layer of
cells on ATR crystal; and
[0239] Measure Cycle. [0240] Begin spectroscopic measurement when
sufficient cell membrane signal exists
[0241] FIG. 12 shows a method 1200 of analyzing a sample. At a step
1210, the sample is acquired as described herein. At a step 1220,
the acquired sample is separated as described herein, for example
with gravimetric separation and washing. At a step 1230, spectra
are measured from the sample and statistical analysis methods can
be used to determine the history of the cell such as the red blood
cell. The analysis methods may comprise one or more of principle
components analysis (PCA), principle components regression (PCR),
multivariate curve resolution (MCR), classical least squares (CLS),
partial least squares regression (PLS), neural networks, or other
biostatistical or chemometric approaches, for example. At a step
1240, a plurality of factors is generated. The factors can be
orthogonal to each other, for example. At a step 1250, one or more
relevant factors is identified. At a step 1260 the red blood cell
history is determined in response to the one or more relevant
factors. At a step 1270, the above steps are repeated.
[0242] FIG. 12 shows a method of analyzing a sample in accordance
with embodiments. A person of ordinary skill in the art will
recognize many adaptations and variations in accordance with the
embodiments disclosed herein. For example one or more steps can be
deleted. Steps can be added, and some steps can be repeated. At
least some of the steps may comprise sub-steps.
[0243] The method 1200 can be embodied with instructions of a
processor on a tangible medium. The processor may comprise one or
more a computer, a cloud computer, a computer network, a digital
processor, a digital signal processor, gate array logic, field
programmable gate array, programmable array logic. The tangible
medium comprises may comprise a storage structure to store
instructions of the processor, for example a computer readable
memory such as flash memory, random access memory or a hard disk
drive.
[0244] The methods and apparatus disclosed herein can be configured
in one or more of many ways to measure vibrational spectroscopy of
the sample, such as infrared (IR) spectroscopy, near infrared
spectroscopy, visible spectroscopy, Raman spectroscopy, nuclear
magnetic resonance (NMR) spectroscopy, total internal reflection
(TIR) spectroscopy, TIR-IR spectroscopy, transmission spectroscopy,
transmission IR spectroscopy, or transmission near-IR
spectroscopy.
[0245] The methods and apparatus disclosed herein can be configured
to determine spectral changes in a blood sample in response to one
or more of drying of the blood sample, washing of the blood sample,
hyper-molality of the blood sample, hypo-molality of the blood
sample, temperature of the blood sample, heating of the blood
sample, cooling of the blood sample, pressure of the blood sample,
pressurization of the blood sample, and depressurization of the
blood sample.
[0246] For example, the methods and apparatus described herein can
be configured to measure spectroscopic data of red blood cells over
a time period of a drying process. The red blood cells may be
purified and washed, e.g., resuspended to 20% hematocrit in
phosphate buffered saline, then subjected to a gradual drying
process, and the sample may be measured spectroscopically as
described herein at regular time intervals. Such a measurement can
provide a study of how the chemical composition, protein structure
and/or conformation of the red blood cell membrane changes over a
drying process. Work in relation embodiments suggests that the
methods and apparatus as described herein may be well-suited for
the measurement of dried blood samples. Without being bound by any
particular theory, the drying of red blood cells can provide some
enhancement in spectroscopic measurements. For example, since water
is known to interfere with infrared measurements, the removal of
water from the sample may improve the spectral signal of the sample
of interest. Alternatively or in combination, the removal of water
from the sample may cause the sample region of interest, e.g., red
blood cell membranes, to adsorb on the measurement surface,
resulting in an improvement of the spectral signal of interest.
Removal of at least some water from the blood samples may further
inhibit lysing of the red blood cells, such that the red blood cell
membranes remain substantially intact during measurement.
Accordingly, the methods and apparatus disclosed herein may be
configured to identify blood pressure of blood samples with at
least some water removed from the blood sample, in order to improve
the spectral signal of the red blood cell membranes. For example,
the blood samples with about 50% of the water of the blood sample
removed may be measured.
[0247] The methods and apparatus as described herein may also be
configured to measure spectroscopic data of blood samples at
different osmolalities, which may cause red blood cells to shrivel,
expand, lyse, or otherwise undergo conformational changes. A
plurality of spectra may be obtained from the blood sample, each
spectra corresponding to a different osmolality.
[0248] The processor as described herein can be configured to
identify a condition of the patient, such as one or more of high
blood pressure or malaria, for example. The processor system can be
configured to analyze the sample as described herein, for example
with one or more of a least squares fit or a classic least squares
fit, for example. Spectral shapes can be associated with blood
pressure, such as mean arterial blood pressure, systolic blood
pressure, diastolic blood pressure, or pulse pressure, for example.
The processor may comprise instructions to identify high blood
pressure of the patient in response to one or more spectral
signatures as described herein, for example by determining a
plurality of spectral factors as described herein
[0249] The methods and apparatus disclosed herein can be used to
identify a condition of a patient in response to spectra of a blood
sample of the patient. The thus-identified condition may be used to
determine an appropriate course of treatment for the patient, such
as to identify a drug to administer to the patient or to determine
the amount of said drug to administer to the patient. For example,
the processor of the apparatus may comprise instructions to
determine an amount of drug to provide to the patient in response
to spectral data of the patient's blood sample. One or more
clinical trials may be conducted to validate the identification of
the course of treatment using spectral measurements of a patient's
blood sample. For example, the amount of drug for administration to
the patient, determined using the measurement of blood spectral
data, may be validated with one or more clinical trials.
[0250] The methods and apparatus disclosed herein may be suitable
for incorporation with clinical trials. For example, a method of
performing a clinical trial to evaluate a safety and/or efficacy of
a treatment with a device and/or drug may comprise using the
measurement apparatus as described herein to measure blood samples
of patients.
[0251] The methods and apparatus can be configured to provide a
differential measurement of the sample, with first spectra measured
without the sample to calibrate the instrument and second spectra
measured with the sample. The calibration measurements can be
obtained with the sample holder placed in the spectrometer and
without the sample.
[0252] The sample can be measured without over fitting the data,
for example.
[0253] While many computation methods can be used as described
herein, classical least squares can be used to fit bands and
functional groups and provide functional group analysis, for
example. Alternatively or in combination, partial least squares
fitting can be used. Known factors such as one or more of water or
water vapor can be added to the sample a priori, for example.
Augmented classical least squares can be used to analyze the
spectral data.
[0254] The methods and apparatus as described herein can be
configured with instructions to provide augmentation of the
calibration space. While the calibration space augmentation can be
performed in one or more of many ways with the factors and
functions methods as described herein, the calibration space
augmentation may comprise one or more of an augmented classical
least squares of the calibration space data, an augmented partial
least square of the calibration space data, or an multivariate
curve resolution of the calibration space data. An iterative fit
can be performed to linearly independent spectral data sets, for
example. A spectral signature can be developed for one or more of
the calibration space data or the blood sample data, for example.
The spectral signature of the calibration space data can be used
for later analysis of the blood sample as described herein, for
example with one or more of partial least squares, augmented
classical least squares, multivariate curve resolution, or other
chemometric approach as described herein, for example.
[0255] FIG. 22 shows a method 2200 of spectral data analysis
suitable for incorporation with embodiments. A variant of Classical
Least Squares (CLS) may be used to build calibration models and
predict blood pressure values based on red blood cell spectra. This
CLS variant has been referred to as Augmented CLS and can often be
performed during the prediction process. CLS assumes Beer's law
behavior (A=CK+E.sub.A), where A is the absorbance spectra, C is a
matrix of concentrations, K is the pure component spectra and
E.sub.A are the spectral residuals (anything unmodelled by linear
combination of C and K). Red blood cell spectra obtained using a
measurement apparatus as described herein can be converted to
absorbance by taking the minus Log 10 of the ratio of the red blood
cell spectra to a close-in-time instrumental background spectrum.
Since CLS tries to minimize E.sub.A, all sources of spectral
variation need to be modelled through the concentrations (C) and
the pure component spectra (K) in order to produce accurate
resultant estimates. The pure component spectrum (K) of an analyte
of interest is usually already known; therefore augmentation
usually occurs in the prediction process (solving for C). To
prevent aberrant spectral variation (spectral variation not
associated with the analyte of interest) from affecting the CLS
model, the model may be proactively augmented with spectral
component(s) associated with these aberrations, so that better
concentration estimates of the analyte of interest can be obtained.
The augmentation process may be applied during the calibration
process, in order to get an accurate estimate of the spectral pure
component associated with blood pressure.
[0256] At step 2202, a concentration matrix C is created to obtain
the pure spectral component of blood pressure. This concentration
matrix can be composed of blood pressure reference measurements
(C.sub.BP), concentrations associated spectral variance during the
measurement of the red blood cell samples but not associated with
the red blood cells (C.sub.S), and concentrations associated with
spectral variance of the instrument (C.sub.I). Concentrations
C.sub.BP, C.sub.S, and C.sub.I can be combined into one
concentration matrix C, and used to estimate the pure spectral
components that can be used for later predictions.
[0257] At step 2204, the blood pressure reference values (C.sub.BP)
are obtained. The blood pressure reference values C.sub.BP may
comprise the mean of the blood pressures acquired over a period of
time from a subject, to ensure the best estimate of the actual
sustained blood pressures from the subject.
[0258] At step 2206, the concentrations associated with spectral
variance during the measurement of the red blood cell samples
(C.sub.S) are obtained.
[0259] At step 2208, previously obtained pure spectral components
(K.sub.S) are applied. Spectral components K.sub.S may comprise
spectral components of water, red blood cells, and spectral
variation associated with a process applied to the red blood cells,
such as drying.
[0260] At step 2210, the concentrations C.sub.S are estimated using
CLS, from the pseudo inverse of the previously obtained pure
spectral components K.sub.S and the absorbance spectra A. The
pseudo inverse K.sup.+ of the spectral components K.sub.S can be
obtained using the equation K.sup.+={circumflex over
(K)}.sub.s.sup.T({circumflex over (K)}.sub.s{circumflex over
(K)}.sub.s.sup.T).sup.-1, where {circumflex over (K)}.sub.s.sup.T
is the transpose of the matrix {circumflex over (K)}.sub.s.
[0261] At step 2212, the concentrations associated with the
instrument variation (C.sub.I) are obtained.
[0262] At step 2214, instrumental background spectra (Bkg) are
applied. Background spectra Bkg may be taken during the entire
period of absorbance spectra (A) data collection. These background
spectra can comprise measurements of air (no sample in sample
compartment of instrument), or measurements of a sample that most
spectrally resembles the sample of interest, but is not the actual
sample of interest (e.g., water or saline). These background
spectra can be decomposed into spectral factors or components
(K.sub.I) by using Principal Component Analysis (PCA). The number
of these spectral components (K.sub.I) can be varied, such that
only the largest sources of spectral variance are explained by
these spectral components (K.sub.I).
[0263] At step 2216, the concentrations associated with the
instrument variation C.sub.I are estimated using CLS, from the
pseudo inverse of the instrument variation spectral components
K.sub.I and the absorbance spectra A. The pseudo inverse K of the
spectral components K.sub.I can be obtained using the equation
{circumflex over (K)}={circumflex over (K)}.sub.I.sup.T
({circumflex over (K)}.sub.I{circumflex over
(K)}.sub.I.sup.T).sup.-1, where {circumflex over (K)}.sub.I.sup.T
is the transpose the matrix {circumflex over (K)}.sub.I.
[0264] At step 2218, the calibration model is built by using a CLS
calculation to obtain the pure component spectra K of which the
component of interest resides, from the pseudo inverse of the
concentration matrix C and absorbance spectra. The pseudo inverse C
of the concentrations C can be obtained using the equation
{circumflex over (K)}=(C.sup.TC).sup.-1C.sup.T where C.sup.T is the
transpose the matrix C. The spectral component of interest can be,
for example, the component associated with blood pressure.
[0265] At step 2220, the concentration C of the component of
interest is predicted using traditional CLS, from the pseudo
inverse of the pure component spectra K and the absorbance spectra
A. The pseudo inverse K of the spectral components K can be
obtained using the equation K.sup.+={circumflex over
(K)}.sup.T({circumflex over (K)}{circumflex over
(K)}.sup.T).sup.-1, where {circumflex over (K)}.sup.T is the
transpose of the matrix {circumflex over (K)}. The concentration C
can be, for example, the blood pressure level. Using this
prediction model, blood pressure may be predicted using spectral
data of blood samples acquired in the future by using traditional
or augmented CLS methods.
[0266] The method 2200 discloses a method of predicting blood
pressure from spectroscopic data from blood samples, in accordance
with embodiments. A person of ordinary skill in the art will
recognize many variations and modifications based on the disclosure
provided herein. For example, some steps may be modified, some
steps may be added or removed, some of the steps may comprise
sub-steps, and many of the steps can be repeated.
[0267] The processor as described herein can be programmed with one
or more instructions to perform one or more of the steps of the
method 2200 of predicting blood pressure using blood spectroscopic
measurements. Therefore, the above steps are provided as an example
of a method of measuring blood pressure of the subject in
accordance with embodiments.
[0268] Work in relation to embodiments suggests that the methods
and apparatus disclosed herein are well suited to determine early
stages of malaria, for example before a ring structure becomes
visible under a microscopic view of a blood sample. As malaria can
induce changes to the red blood cell membrane, the spectroscopic
analysis of the red blood cell membrane as described herein can be
used to identify malaria.
[0269] The spectrometer as described herein may comprise a hand
held portable spectrometer for example. The spectrometer may
comprise an optical window that can be wiped off subsequent to
measurement of the blood sample, and used repeatedly with cleaning,
for example. Alternatively, the spectrometer may comprise a
consumable single use window component as described herein, for
example.
EXPERIMENTAL
[0270] Based on the teachings disclosed herein, a person of
ordinary skill in the art can identify biomarkers in blood in order
to determine the presence of hypertension. A person of ordinary
skill in the art can conduct experiments to identify one or more
additional biomarkers in order to predict current and/or recent
central aortic vessel pressures.
[0271] The apparatus can be constructed as described herein to
measure the one or more biomarkers of the blood sample. A
population of subjects can be measured with the apparatus to
determine the presence of biomarkers and this data can be compared
with measured blood pressure of the subjects. The relationship
among the one or more biomarkers and blood pressure can be
determined with one or more analytic models as described herein.
For example, the high blood pressure of the subject can be
identified in response to the amount of biomarker measured, and the
high blood pressure can be presented to the physician as one or
more of an index or a scale. In many embodiments, the amount of
biomarker can be mapped to a traditional systolic blood pressure
with a mapping function such as a look up table or scaling factor.
For example, the systolic blood pressure can be determined with a
linear function such as
BLOOD PRESSURE=A*[CONCENTRATION OF BIOMARKER]+B
[0272] where the BLOOD PRESSURE is the determined blood pressure in
mm Hg in response to the CONCENTRATION OF BIOMARKER in ng/ml times
the scaling constant A plus the offset constant B. The parameters A
and B can be determined based on the study population, for
example.
[0273] FIG. 13 shows a commercially available spectroscopy
apparatus 1300 suitable for combination in accordance with
embodiments. The commercially available spectroscopy apparatus may
comprise an ALPHA-P spectrometer, and may comprise an evanescent
wave FT-IR spectrometer for example. The commercially available
evanescent wave spectrometer can be used to measure one or more
model substances 1310 such as chicken red blood cells, fresh, or
treated with gluteraldehyde to stiffen the membrane, for
example.
[0274] FIG. 14 shows example spectra of fat 1400, milk 1410, dried
red blood cells 1420, red blood cells 1430, red meat 1440, and red
wine 1450.
[0275] FIG. 15 shows an aspirin study. The aspirin study shows
principal component analysis components eigen vector 1 1510, eigen
vector 2 1520, and eigen vector 3 1530. Aspirin study shows a human
subject's response to a baby aspirin. The study used the first 10
spectra from each data set. PCA shows a difference in the blood
sample without aspirin 1500 and blood sample with aspirin 1505. The
first factor 1510 corresponds to intensity differences in the
signal. The second factor 1520 corresponds to a change because of
the shift in the Amide II peak (positive for the no aspirin samples
and negative for the aspirin samples).
[0276] FIG. 16A shows multivariate curve resolution (MCR) factors.
Factor 1 may comprise spectral peaks such as one or more of a
carboxylate peak 1600, a CH3 bending peak 1605, an Amide II peak
1610, or an Amide I peak 1615, for example. Factor 3 may comprise
Amide I, Amide II broadening 1620, for example. Factor 4 may
comprise a water peak 1625, for example, at 1560 cm.sup.-1 (inverse
centimeters). Factor 5 may comprise a 1560 cm.sup.-1 shift. Factor
6 may comprise a baseline offset, for example. Many additional
factors can be used in accordance with the embodiments described
herein, for example.
[0277] FIG. 16B shows MCR concentrations for the factors of FIG.
16A for chicken blood preliminary results as follows:
[0278] 1-5: Fresh Supernatant
[0279] 6-10: Gluteraldehyde Supernatant
[0280] 9-20: Fresh Cells, 3 replicates at each settling time (time
0: F1,F3,F5; time 1: F1,F3,F5; time 2: F1,F3,F5; time 3:
F1,F3,F5)
[0281] 21-32: Glut Cells, 3 replicates at each settling time (time
0: G2,G4,G6; time 1: G2,G4,G6; time 2: G2,G4,G6; time 3:
G2,G4,G6)
[0282] These preliminary data show concentration differences among
the samples in accordance with embodiments described herein.
[0283] The analysis may comprise one or more analysis tools of
commercially available software such Chemometrics metrics software
available from Eigen Vector Research Incorporated, for example as
listed on with World Wide Web
(www.eigenvector.com/software/solo.htm). The software may comprise
one or more of the following capabilities: [0284] Data Exploration
and Pattern Recognition (Principal Components Analysis (PCA),
Parallel Factor Analysis (PARAFAC), Multiway PCA) [0285]
Classification (soft independent modeling of class analogies
(SIMCA), k-nearest neighbors, Partial Least Squares (PLS)
Discriminant Analysis, Support Vector Machine Classification,
Clustering (Hierarchical Cluster Analysis, HCA)) [0286] Linear and
Non-Linear Regression (PLS, Principal Components Regression (PCR),
Multiple Linear Regression (MLR), Classical Least Squares (CLS),
Support Vector Machine Regression, N-way PLS, Locally Weighted
Regression) [0287] Self-modeling Curve Resolution, Pure Variable
Methods (Multivariate Curve Resolution (MCR), Purity (compare to
SIMPLSMA), CODA_DW, CompareLCMS) [0288] Curve fitting and
Distribution fitting and analysis tools [0289] Instrument
Standardization (Piece-wise Direct, Windowed Piece-wise, OSC,
Generalized Least Squares Preprocessing) [0290] Advanced Graphical
Data Set Editing and Visualization Tools [0291] Advanced
Customizable Order-Specific Preprocessing (Centering, Scaling,
Smoothing, Derivatizing, Transformations, Baselining) [0292]
Missing Data Support (Singular Value Decomposition (SVD) and
Non-Linear Iterative Partial Least Squares (NIPALS)) [0293]
Variable Selection (Genetic algorithms, Iterative PLS (IPLS),
Selectivity, Variable Importance Projection (VIP))
[0294] FIG. 17 shows results from a study with
gluteraldehyde-treated red blood cells. A scatter plot of MCR
Factor 3 in relation to Factor 4 is shown. The data are shown for
combined settling times of 2, 4, and 6 minutes. Membrane secondary
structural changes can be induced by brief treatment with
gluteraldehyde. Washed intact chicken red blood cells were
obtained, some fresh and some treated briefly with gluteraldehyde.
Membrane secondary structural changes are clearly visible based on
the comparison of Factor 3 and Factor 4.
[0295] Gluteraldehyde induces structural changes in the red blood
cell membrane and is capable of denaturing proteins. Without being
bound by any particular theory, the spectral changes induced by
gluteraldehyde can have at least some similarity to spectral
changes induced by blood pressure of the subject. For example, the
red blood cell of the hypertensive subject can be more deformable
than a subject having normal blood pressure. Gluteraldehyde is a
cross-linking molecule that affects the structural rigidity of the
red blood cell membrane.
[0296] FIG. 18 shows results from a study with human blood and
aspirin. Whole blood from one volunteer was obtained via
fingerstick before and after the ingestion of acetylsalicylic acid
(ASA, aspirin). Aspirin induces membrane structural changes in the
red blood cell. A drop of heparinized blood was measured directly
on a horizontal sampler and spectra were acquired while allowing
the red blood cells to gravimetrically separate from whole blood
and deposit onto the sampler window. This was done to allow
chemometric separation of the pure membrane spectrum. Data were
analyzed using multivariate curve resolution (MCR). This experiment
was repeated 4 times on 4 separate days, and the data set consists
of 80 full infrared spectra. The data for MCR factor 6 and factor
10 show a clear separation between red blood cell membrane before
and after ingestion of aspirin. Results are consistent across all 4
study days.
[0297] FIG. 19 shows MCR factors 3, 6, and 10, in accordance with
embodiments. Factor 3 may correspond to the protein structure of
blood. Factor 3 can be used as a reference for two or more factors
that allow discrimination of blood after an oral dose of aspirin.
Factor 6 may correspond to a shift in the Amide I peak. Factor 10
may correspond to a shift in Amide II, for example.
[0298] FIG. 20 shows a 3D plot of results from a study of the
effect of gluteraldehyde on blood.
[0299] FIG. 21 shows a 2D plot of the data of FIG. 20. Factor 3
represents the protein structure of the blood and is used as a
reference for factors 6 and 10. Factor 6 predominantly exhibits a
shift in Amide I. Factor 10 predominantly exhibits a shift in Amide
II.
[0300] Spectra were taken on an untreated blood sample and the last
ten equilibrated spectra 2000 were selected for use in the further
analysis. Blood was treated with gluteraldehyde and spectra were
taken, with the last ten equilibrated spectra 2010 being selected
for use in the further analysis. Spectral data were normalized to
the Amide I peak 1610. Changes from after the gluteraldehyde
treatment include a small shift in the Amide I peak, a larger shift
in the Amide II peak 1615, a change in Carboxylate peak 1600
intensity, and an increase in Amide III band 1630.
[0301] FIG. 23 shows results from a study of mean arterial blood
pressure (MAP) measurements in human subjects using a
sphygmomanometer or blood pressure cuff. Blood pressure was
monitored in 11 subjects, 8 having high blood pressure (systolic
140-170 mmHg/diastolic 90-120 mmHg) and 3 having normal to low
blood pressure, over a period of 28 days. Subjects were trained in
the use of an ambulatory blood pressure (ABP) monitoring device
(Welch Allyn ABPM6100 Blood Pressure Monitor), designed for 24-hour
blood pressure monitoring. Subjects recorded blood pressure
readings once every day for 5 days of a week, and 6 times a day for
2 days of a week. FIG. 23 shows the MAP values (mmHg) averaged per
subject, wherein the MAP values were calculated from the systolic
(SP) and diastolic (DP) cuff measurements using the equation
MAP=((2.times.DP)+SP)/3. In FIG. 23, each vertical "line" of data
points represents MAP measurements for a single subject, and the
sloped line going through all of the vertical "lines" of data
points shows the average of the MAP measurements for each subject
over the 28-day study period. The data shows the wide variation in
the cuff measurements for each subject over the study period. Any
one of the data points in each vertical "line" of data points may
represent a single cuff measurement taken from a patient, and as
shown in FIG. 23, a single cuff measurement may be significantly
different from the average MAP value for the patient over the
28-day study period (data point through which the sloped line
extends). It is generally recognized that the average level of
blood pressure over prolonged periods of time represents the
measure of blood pressure that is most clearly related to morbid
events in patients. However, clinic measurement often comprise
single cuff readings taken in the office, and ambulatory blood
pressure (ABP) monitoring are not widely used because the devices
may be cumbersome and inconvenient for patients. The results of the
study in FIG. 23 show that single cuff readings can often be
inaccurate in determining the patient's true average blood pressure
over a prolonged period of time.
[0302] FIG. 24 shows results from a study of mean blood pressure
measurements in human subjects using a measurement apparatus in
accordance with embodiments. Blood samples were drawn once a day
over the 28-day study period from the same 11 subjects as in the
study of FIG. 23. The blood samples were analyzed using the
measurement apparatus as described herein, using TIR spectroscopy
to measure changes in the membrane of the red blood cells in the
blood samples. Subsequently, spectroscopic data was analyzed using
Augmented Classical Least Squares methods as described herein. The
analyzed spectroscopic data was converted to predicted blood
pressure values (mmHg) using a prediction function as described
herein. FIG. 24 shows the predicted mean arterial pressure (MAP)
values (mmHg) derived from spectroscopic measurements of blood
samples for each subject over the 28-day study period, such that
each vertical "line" of data points represents predicted MAP values
from blood measurements from a single subject. The predicted MAP
values are graphed against reference MAP measurements, derived from
cuff measurements as described for FIG. 23, and the sloped line
going through the data points shows the average of the MAP
measurements for each subject over the 28-day study period. FIG. 24
shows that the predicted MAP values derived from blood
spectroscopic measurements are able to predict the average MAP
measurements with a standard error of about 11.7 mmHg, and a
coefficient of determination (R.sup.2) of about 0.7. Comparing the
results of FIG. 24 with the results of FIG. 23, it can be seen that
a single blood spectroscopic measurement, represented by a single
data point in each vertical "line" of data points, can more closely
predict the average MAP value for a patient over the 28-day study
period (data point through which the sloped line extends) than a
single cuff reading, represented by a single data point in each
vertical "line" of data points in FIG. 23. The accuracy of
predicting average blood pressure values using blood spectroscopic
measurements may be further improved by appropriate modifications
to the measurement apparatus and/or data analysis algorithms. While
FIG. 24 shows mean arterial pressure values, the spectroscopic data
can be converted to systolic blood pressure, diastolic blood
pressure, pulse pressure, or any other clinically relevant measure
of blood pressure. It is noted that for the results of one of the
subjects shown in FIG. 24, in-clinic mercury sphygmomanometer
measurements were for substituted for the subject-provided ABP
measurements in deriving the reference MAP measurements, because
the ABP monitoring device used by the subject was found to be
functioning improperly during the course of the study.
[0303] FIG. 25 shows additional results from the study of FIG. 24.
The study was conducted in 11 human subjects, 8 having high blood
pressure (systolic 140-170 mmHg/diastolic 90-120 mmHg;
"hypertensive") and 3 having normal to low blood pressure
("normal"). The predicted mean arterial pressure (MAP) values
(mmHg) derived from blood sample spectroscopic measurements were
averaged per subject group ("normal" and "hypertensive"). In FIG.
25, the center line of each box plot represents the median of the
MAP values for each subject group, while the top and bottom of the
boxes represent 25.sup.th and 75.sup.th percentiles. FIG. 25 shows
that for predicted MAP values obtained from blood spectroscopic
measurements, the median MAP of the "normal" subject group is found
to be statistically different from the "hypertensive" subject
group, with 95% confidence.
[0304] Work in relation with embodiments suggests that the methods
and apparatus as described herein may be well-suited for the
measurement of blood samples that have been stored up to about 3
days after collection. No significant changes in spectroscopic data
of blood samples were observed during such a time window, when red
blood cells were purified, washed, and stored under appropriate
refrigeration conditions.
[0305] Spontaneously Hypertensive Mouse and Rat Studies
[0306] Work in relation to embodiments suggests that animal models
can be used to identify biomarkers suitable for use in humans.
Vertebrate erythrocytes consist mainly of hemoglobin. The mammalian
red blood cell comprises similar structures, proteins, and
biomarkers among many species including mammals such as humans,
rats, and mice. Mammalian erythrocytes typically have a biconcave
disk shape, which optimizes their flow properties in larger
vessels. Generally, mammalian erythrocytes are flexible and
deformable to enable passage through small capillaries.
[0307] Mammalian erythrocytes are non-nucleated in their mature
form, and also lack all other cellular organelles. Consequently,
they lack DNA and cannot synthesize RNA. Structural properties are
linked to the membrane. The membrane comprises a lipid bilayer,
membrane proteins, lipids, and carbohydrates. The membrane is
composed of three layers: the outer, carbohydrate-rich glycocalyx,
the lipid bilayer, and the membrane skeleton. Mammalian erythrocyte
lipid bilayers contain similar compositions of phospholipids,
including choline phospholipids (CPs), acidic phospholipids (APs),
and phosphatidylethanolamine (PE).
[0308] Spontaneously hypertensive rats, and similar model mice,
comprise attributes that can be suitable for identification of
blood markers of health of humans, in accordance with
embodiments.
[0309] High-density lipoprotein (HDL) and low-density lipoprotein
(LDL) are both present in humans, mice, and rats. Wild-type mice
are usually resistant to lesion development and clear LDL very
quickly. Mouse models more useful for comparison to humans have
been developed. For example, low-density lipoprotein
receptor-deficient mice (LDLR-/- mice) and apolipoprotein
E-deficient mice (apoE-/- mice) are widely used. LDLR-/- mice
respond effectively to peroxisome proliferator-activated receptor
(PPAR) agonists, which are used in humans as well to reduce
triglycerides (TG) and LDL cholesterol and to raise HDL
cholesterol. ApoE-/- mice develop extensive atherosclerotic
lesions, and respond to treatment with statins and PPAR agonists,
as do humans.
[0310] The spontaneously hypertensive rat (SHR) is another animal
model of primary hypertension commonly used to study cardiovascular
disease. Around 5-6 weeks of age, the SHR begins hypertensive
development. In adult age, systolic pressures reach 180-200 mmHg.
Around 40-50 weeks, the SHR typically develops characteristics of
cardiovascular disease, such as vascular and cardiac hypertrophy.
Similar models have been developed in mice, such as JAX BPL/2 mice.
BPL/2 mice develop elevated systolic blood pressure at five weeks
of age, and by 150 days of age show an average blood pressure of
119 mmHg. This predictable progression allows longitudinal studies
of the same population both before and after hypertensive
development. Such studies can show biomarker levels and other
changes associated with the onset of hypertension and/or the
impacts of hypertension.
[0311] With the teachings of the present disclosure, a person of
ordinary skill in the art can conduct experiments to measure and
identify blood based biomarkers to determine the health of a human
subject without undue experimentation.
[0312] Reference is made to the following claims which recite
combinations that are part of the present disclosure, including
combinations recited by multiple dependent claims dependent upon
multiple dependent claims, which combinations will be understood by
a person of ordinary skill in the art and are part of the present
disclosure.
[0313] While preferred embodiments of the present disclosure have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
be apparent to those skilled in the art without departing from the
scope of the present disclosure. It should be understood that
various alternatives to the embodiments of the present disclosure
described herein may be employed without departing from the scope
of the present invention. Therefore, the scope of the present
invention shall be defined solely by the scope of the appended
claims and the equivalents thereof.
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