WO2011130304A2

WO2011130304A2 - Materials and methods for reliable measurement of blood volume

Info

Publication number: WO2011130304A2
Application number: PCT/US2011/032158
Authority: WO
Inventors: Nathan J. Rice; Lennox Hoyte; Jianfeng Cai
Original assignee: University Of South Florida
Priority date: 2010-04-12
Filing date: 2011-04-12
Publication date: 2011-10-20
Also published as: WO2011130304A3

Abstract

The present invention concerns materials and methods for measuring the blood volume in a human or animal subject. The method of the invention involves determining the blood volume in the vasculature of an individual by determining the amount of detectable label in a sample of blood obtained from the individual after a volume of infusion media containing a known amount of the label has been introduced and allowed to be distributed and equilibrate within the individual's vascular system.

Description

DESCRIPTION

MATERIALS AND METHODS FOR RELIABLE MEASUREMENT

OF BLOOD VOLUME

CROSS-REFERENCE TO RELATED APPLICATION The present application claims the benefit of U.S. Provisional Application Serial No. 61/323,217, filed April 12, 2010, which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, and drawings.

BACKGROUND OF THE INVENTION

Blood volume, or intravascular volume status, is the amount of blood present in an individual's circulatory system. Blood volume measurement data can be used in a variety of medical and research contexts in assessing the health of a subject. In many situations, such as during or after surgery, traumatic accident or in disease states, it is desirable to identify and quantify the amount of blood loss the patient has suffered, to determine the percentage of red blood cells or hemoglobin the patient has lost, to restore a patient's blood volume to normal as quickly as possible, and to assess the need for continuing treatment.

Blood volume is related to an individual's state of hydration. Deviations from normal blood volume (normovolemia) can have adverse clinical effects. Intravascular blood volume depletion is termed hypovolemia. Signs of hypovolemia include a fast pulse, infrequent and low volume urination, dry mucous membranes, poor capillary refill, decreased skin turgor, weak pulse, orthostatic hypotension, and cool extremeties. Hypovolemmia can be caused by dehydration, bleeding, vomiting, severe burns, and drugs such as diuretics or vasodilators, and rarely by blood donation, sweating, and alcohol consumption. Intravascular blood volume overload or hypervolemia can occur due to disorders of the heart, kidneys or lungs. Unrecognized hypervolemia may contribute to worsening symptoms and disease progression in patients with chronic heart failure (CHF) (Androne A.S. et al, Am. J. Cardiol, 2004, 93: 1254-1259. which is incorporated herein by reference in its entirety). Symptoms of hypervolemia and increased cardiac filling pressures in the systemic and pulmonary venous circulations are among the most common complaints in patients with CHF ( atz, S.D., Am. J. Med. Sci. , 2007, 334(1 ):47-52, which is incorporated herein by reference in its entirety). Increased blood volume in heart failure results from a complex interaction of hemodynamic and biomolecular factors that induce renal sodium and water retention in response to decreased cardiac output and renal hypoperfusion.

Blood volume has typically been measured indirectly by evaluating multiple parameters. Many clinicians use a combination of clinical assessment and surrogate tests to indirectly determine blood volume. Clinical assessment relies on indirect indicators such as blood pressure, weight change, lung sounds, edema, and jugular venous distension. Surrogate tests such as hematocrit and hemoglobin only measure the ratio of red blood cells to total blood volume. Highly invasive tests like the pulmonary capillary wedge pressure measure pressures in the heart and lung. Although these tests offer useful clinical information, they do not measure blood volume directly. Moreover, these indirect measures are not as accurate or reliable as direct methods of measuring blood volume due to inter- and intra-subject variability in measurements based on these parameters. Direct blood volume measurement can be much more accurate in providing complementary information that can improve a patient^'s clinical outcome.

Direct measurement is conventionally accomplished by the "indicator dilution technique" involving: (i) drawing a volume of the patient's blood; (ii) separating the blood components; (iii) attaching an indicator or tag. such as a radioactive isotope or chemical dye, to either the plasma protein portion of the plasma or the red cell portion of the patient^'s blood; (iv) reinjecting the tagged red cells or plasma protein into the patient's blood stream; (v) waiting for the tagged red cells or plasma protein to equilibrate within the patient's vascular system; (v) drawing another volume (sample) of the patient^'s blood; and measuring the concentration of the tag in the blood sample and computing the blood volume. The degree of dilution of the tag is inversely related to the volume of the patient's blood. Through chemical analysis or use of a gamma counter, the degree of dilution of the tag is ascertained and mathematically related to the absolute measurement of the patient's blood volume. This represents a patient-specific process (i.e., requiring an initial blood draw) that is time consuming and impractical for routinely measuring blood volumes in large populations. BRIEF SUMMARY OF THE INVENTION

The present invention concerns materials and methods for measuring the blood volume in a human or animal. The method of the invention involves determining the blood volume in the vasculature of an individual by determining the amount of detectable label in a volume of blood obtained from the individual after a volume of fluid comprising a known (pre-determined) amount of the detectable label (also referred to herein as labeled infusion media) has been introduced and allowed to equilibrate within the individual's vascular system.

The infusion media is a non-blood fluid that can be labeled with a detectable substance and is not susceptible to extravascular leakage, e.g., having molecules that are sufficiently large and/or having an appropriate net charge so as not to leak out through the blood vessel walls and be retained in the intravascular space, and /or having molecules that bind to components of the subject's blood before extravascular leakage can occur. Preferably, the infusion media introduced into the individual's vascular system is a volume expander or oxygen therapeutic (also known as a blood substitute or synthetic blood product).

Unlike conventional indicator dilution techniques, the method of the invention can be conducted with a single injection, with a single label, and a single blood draw. Furthermore, the labeled infusion media can be pre-mixed, allowing stocking and storage for future use. The method of the invention is therefore patient-independent (i.e., does not require the patient's blood to be drawn in order to initiate the method). Accordingly, the labeled infusion media can be mass-produced and mass-implemented. Unlike an albumin-based measurement, the infusion media utilized in the method of the invention does not leak into the extravascular space; therefore, the blood volume measurement does not have to be corrected for leakage (i.e., multiple blood draws are not required to assess extravascular leakage).

In some embodiments, the method for measuring the blood volume in a human or animal subject comprises:

(a) introducing a volume of infusion media comprising a pre-determined amount of a detectable label (labeled infusion media) into the vascular system of the subject;

(b) allowing the volume of labeled infusion media to equilibrate within the vascular system (but not be eliminated):

(c) obtaining a volume of blood (sample) from the vascular system; and (d) determining the blood volume in the vascular system based on the amount of label in the volume of blood.

The degree of dilution of the label is inversely related to the volume of the subject's blood. Through the use of a detection system (such as a scintillation counter in embodiments in which the label is radioactive), the degree of dilution of the label can be ascertained and mathematically related to the absolute measurement of the subject's blood volume.

In some embodiments, the infusion media is an oxygen therapeutic (also known as an intravascular oxygen carrier) such as a periluorocarbon (PFC) compound or hemoglobin- based oxygen carrier (HBOC). In some embodiments, the fluid is a volume expander such as a dextran solution, saline solution {e.g., sodium chloride), Ringer's solution, hetastarch (also known as hydroxyethyl starch), or pentastarch. In some embodiments, the infusion media is a PFC or PFC derivative.

In some embodiments, the HBOC is a composition comprising polymerized hemoglobin. In some embodiments, the HBOC is Hemoglobin Gutamer-250 (bovine), which is also known as HBOC-201 and Hcmopure® (Biopure Corp.. Cambridge. MA). In some embodiments, the oxygen therapeutic is a PFC compound formulated as an emulsion (a PFC emulsion). In some embodiments, the oxygen therapeutic is an emulsion of a PFC, water, salts, and a surfactant, such as the perilubron emulsion Oxygent (Alliance Pharmaceutical Corp., San Diego, CA).

The molecules of the infusion media can be labeled with any detectable moiety that is pharmaceutically acceptable (non-toxic at the desired amounts and compatible with the physiology of the subject). In some embodiments, the label is a radionuclide. In some embodiments, the label is iodine- 129, iodine- 13 1 , or chromium-51. In some embodiments, the detectable moiety is a fluorescent dye. In some embodiments, the detectable moiety is a fluorescent dye having absorption and emission wavelengths in the near-infrared (NIR) spectrum, between 680 ran and 800 nm. In some embodiments, the detectable moiety is a functional derivative of an infrared dye that is reactive toward free -SH groups ( e.g., IRDye 800CW Malcimide). In some embodiments, the infusion media is a PFC or PFC derivative conjugated to a fluorescent dye.

Preferably, the labeled infusion media is intravascularly introduced into the subject' s circulation via an intravascular delivery device, such as a syringe. In some embodiments, the volume of labeled infusion media that is introduced {e.g., intravascularly injected) into the subject is 2-3 cc. Following introduction of the labeled infusion media into the subject's vascular system, a sufficient period of time is allowed to elapse before the blood sample is obtained from the subject so that the labeled molecules of the infusion media will circulate, mix, and be distributed (equilibrate) throughout the subject's circulatory system. In some embodiments, the sample of blood is obtained from the subject at least 30 minutes after the labeled infusion media is introduced. In some embodiments, the sample of blood is obtained from the subject 30 minutes to 60 minutes after the labeled infusion media is introduced.

In some embodiments, the sample of blood obtained from the subject is 5 cc to 10 cc. Another aspect of the invention is directed to a composition comprising labeled infusion media that may be used in carrying out the methods of the invention. In some embodiments, the labeled infusion media comprises an oxygen therapeutic selected from a perfluorocarbon (PFC) compound or hemoglobin-based oxygen carrier (HBOC). In some embodiments, the label is a radionuclide. In some embodiments, the label is iodine- 129. iodine-131 , or chromium-51 . In some embodiments, the labeled infusion media comprises an emulsion of PFCs, water, salts, and a surfactant, labeled with iodine- 13 1 . In some embodiments, the labeled infusion media comprises Hemoglobin Gutamer-250 (bovine) labeled with chromium-51. In some embodiments, the label is a fluorescent dye having absorption and emission wavelengths in the near-infrared (NIR) spectrum, between 680 nm and 800 nm. In some embodiments, the label is a functional derivative of an infrared dye that is reactive toward free -SH groups (e.g., IRDye 800CW Maleimide). In some embodiments, the infusion media is a PFC or PFC derivative conjugated to a fluorescent dye.

Another aspect of the invention comprises an intravascular delivery device, such as a syringe for intravascular injection, containing infusion media and a label, which may be used in carrying out the methods of the invention. Syringes typically include a chamber or reservoir for holding the fluid to be delivered and a plunger to inject the fluid out of the chamber. Syringes of the invention can include a needle or be adapted to accept a needle prior to use. In some embodiments, the delivery device comprises a chamber containing a composition of the invention in which the infusion media and label are components of the same formulation, e.g., pre-mixed. In some other embodiments, the delivery device comprises a first chamber and a second chamber containing the infusion media and label, respectively, and wherein the first chamber and the second chamber are separated by a breakable or removable barrier that, upon breaking or removal prior to delivery of the infusion media, establishes fluid communication between the first and second chambers and permits mixing contact between the infusion media and the label.

Another aspect of the invention comprises a kit for blood volume determination, comprising infusion media and a detectable label. The infusion media may be labeled (pre- labeled) or be separate from the label and intended to be mixed prior to use. Optionally, the kit of the invention further comprises printed instructions for carrying its use (e.g. , for carrying out the methods of the invention). In some embodiments, the kit of the invention further comprises an intravascular delivery device, such as a syringe, for administering the labeled infusion media to a subject. Optionally, the intravascular delivery device is preloaded with the infusion media (labeled or unlabeled). In some embodiments, the delivery device comprises a chamber containing a composition of the invention in which the infusion media and label are components of the same formulation, e.g. , pre-mixed. In some other embodiments, the delivery device comprises a first chamber and a second chamber containing the infusion media and label, respectively, and wherein the first chamber and the second chamber are separated by a breakable or removable barrier that, upon breaking or removal prior to delivery of the infusion media, establishes fluid communication between the first and second chambers and permits mixing contact between the infusion media and the label.

Another aspect of the invention is an article of manufacture useful for determining the blood volume in the vasculature of an individual. The article of the invention comprises computer-executable instructions embodied in a computer-readable medium for performing the blood volume determination method described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the chemical structure of perfubron (perfuoro-octyl bromide), a bromine-substituted fluorocarbon.

Figure 2 shows the perfluorocarbon 3, 3,4,4, 5,5, 6,6,7, 7,8,8, 8-Tridecafluoro-l- octanethiol (also known as 1 H. 1 H2H,2H-Perfluorooctanethiol; CAS : 34451 -26-8).

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention involves determining the blood volume in the vasculature of an individual by determining the amount of label in a volume of blood obtained from the individual after a volume of fluid comprising a known (pre-dctermined) amount of a detectable label (also referred to herein as labeled infusion media) has been introduced and allowed to equilibrate within the individual's vascular system. The method of the invention may be used to detect hypervolemia or hypovolemia in a subject.

(b) allowing the volume of labeled infusion media to equilibrate within the vascular system (but not be eliminated);

(c) obtaining a volume of blood (sample) from the vascular system; and

(d) determining the blood volume in the vascular system based on the amount of label in the volume of blood. A standard or control used in a method of the invention may correspond to a blood volume, or label signal representative of a blood volume, obtained from samples of healthy control subjects (e.g., having the same height, weight, and/or gender as the subject in question), from subjects with benign disorders, subjects known to have disorders that affect blood volume, or from samples previously obtained from the subject, or other reference samples. Increased blood volume as compared to a normal standard may be indicative of hypervolemia. Decreased blood volume as compared to a normal standard may be indicative of hypovolemia. In some embodiments, the method of the invention further comprises treating the subject after blood volume has been determined. Subjects determined to have hypovolemia can be treated, for example, by fluid therapy (e.g. , administering fluids, fluid infusion, blood transfusion). Subjects determined to have hypervolemia can be treated, for example, by therapeutic phlebotomy, ultrafiltration, and/or administration of diuretics.

Blood volumes for control samples from healthy subjects may be established by prospective and/or retrospective statistical studies. Healthy subjects who have no clinically evident hypovolemia or hypermolemia, or disease or abnormalities, may be selected for statistical studies. Diagnosis may be made by a finding of statistically different blood volume, or label signal representative of blood volume, compared to a control blood volume or signal, or previous blood volume or signal from the same subject. Blood volume determination can assist a clinician in diagnosis of abnormalities (e.g. , blood volume abnormalities), including those that may not be apparent from a physical exam or other lab tests. Blood volume determination may help to clarify the optimal treatment for congestive heart failure, hypertension, anemia (e.g. , in cancer patients or HIV-positive patients on chemotherapy), s yncop e/ orth st ati e hypotension, dialysis, chronic fatigue, pre- /post surgical testing (e.g., for low blood volume), and septic shock.

Optionally, multiple blood samples can be obtained from the subject, e.g. , at various time points in order to make multiple blood volume determinations over time. For example, the subject may be treated with one or more interventions or test compounds under evaluation between blood sampling. The effect of the intervention on the subject's blood volume may then be determined.

Optionally, the method of the invention can further comprise determining the percentage of red blood cells and/or plasma in the same blood sample from which the blood volume is determined or from a different blood sample obtained from the subject. Optionally, the method of the invention can further comprise determining the amount of red blood cells and/or plasma in the same blood sample from which the blood volume is determined or from a different blood sample obtained from the subject. The percent and/or relative amount of red blood cells and/or plasma in the subject can then be extrapolated.

The infusion media is a non-blood fluid that can be labeled with a detectable substance and is not susceptible to cxtravascular leakage through active or passive mechanisms. Because the infusion media docs not leak into the extravascular space, the blood volume measurement does not need to be adjusted for leakage (i.e., no additional blood samples are required in order to assess leakage). Molecular size, charge, and structure can influence penetration and transport of solute molecules across the vascular endothelial lining (Vink H. and Duling B.R., Am. J. Physiol. Heart Ore. Physiol., 2000, 278( 1 ):H285-H289; van Haaren P.M.A. et al., Am. J. Physiol. Heart Circ. Physiol , 2003, 285:H2848-H2856, which are incorporated herein by reference). Substances that are not susceptible to cxtravascular leakage and thus retained in the vascular lumen may. for example, comprise molecules that are sufficiently large so as not to leak out through the blood vessel walls (i.e., meeting a minimum size threshold for vascular retention by the subject), and/or comprise molecules with an appropriate net charge so as to be retained in the intravascular space, and/or comprise molecules that bind to components of the subject^'s blood before extravascular leakage can occur. The labeled infusion media is retained in the vascular lumen for a period of time sufficient for dilution and equilibration of the label throughout the vascular system and subsequent blood sampling for determination of the subject's blood volume. Infusion media molecules may themselves be detectable by detection systems without the need for coupling of additional detectable moieties and therefore function inherently as a label. Preferably, the infusion media introduced into the individual's vascular system is a volume expander or oxygen therapeutic ( also known as a blood substitute or synthetic blood product).

Volume Expanders

In some embodiments, the infusion media comprises one or more volume expanders.

Non-blood volume expanders, which do not contribute to the oxygen-carrying capacity of blood, are commercially available. They can be very useful in maintaining blood volume and pressure in acute blood loss situations where the remaining amount of red blood cells are sufficiently numerous to provide adequate oxygen delivery to the tissues of the body.

Dextrans are large molecules composed of chains of sugar molecules. They are formed by bacteria and yeast for food storage and are commonly found in dental plaque. In purified form, certain dextrans can be used intravenously (IV) to expand a patient's blood volume in medical emergencies where blood pressure drops to dangerously low levels. They are "osmotically active,^" meaning they hold water within blood vessels and help maintain blood pressure. Dextran solutions may be helpful to maintain blood pressure in a patient with acute bleeding, their basic action is to dilute the remaining red blood cells by retaining more fluid (plasma) within the blood vessels of the body. In doing so, they may improve circulation, but they are not capable of providing additional oxygen-carrying capacity.

Saline and Ringer's solutions are also used to expand blood volume in medical emergencies with very low blood pressures. Saline is essentially salt water, in which the concentration of salt (e.g., sodium chloride) is adjusted to be the same as that normally found in the blood stream. Ringer's solution is similar, but also contains potassium and calcium in physiologic concentrations along with sodium and chloride. Both products act in the same manner to expand and dilute the blood volume when given by IV. Neither increases oxygen- carrying capacity, and both pass freely out of the blood vessels into the surrounding tissues, so their effect on maintaining blood volume is transient and not sustained. They are eventually lost in the urine, but may cause tissue swelling (edema), especially when kidney function is compromised. Hetastarch, also known as hydroxyethyl starch, and pentastarch arc solutions of large molecules composed of many smaller sugar molecules. Their action is essentially the same as that of dextrans, in that they hold water within the blood vessels and, thereby, expand the blood volume and help maintain blood pressure. As with other non-blood volume expanders, hetastarch and pentastarch do not contribute to the blood's oxygen-carrying capacity. In some embodiments, the infusion media does not comprise a volume expander.

Oxygen Therapeutics

In some embodiments, the infusion media comprises one or more oxygen therapeutics. In some embodiments, the oxygen therapeutic is a hemoglobin-based oxygen carrying solution (HBOC). In some embodiments, the HBOC is a composition comprising polymerized hemoglobin. In some embodiments, the HBOC is Hemoglobin Gutamer-250 (bovine), which is also known as HBOC-201 and Hemopure® (Biopure Corp., Cambridge, MA). This solution is made of chemically stabilized, cross-linked bovine hemoglobin in a salt solution. Hemoglobin is normally toxic to the kidneys. In order to detoxify the hemoglobin without compromising its therapeutic usefulness, it is stabilized. Stabilization can be achieved through a number of methods. In Hemopure®, the hemoglobin is stabilized by cross-linking the two alpha and two beta subunits, which stabilizes the alpha-beta dimers. This makes the hemoglobin more stable and reduces the affinity for oxygen. Hemopure® is smaller in size and has less viscosity than human red blood cells, enabling it to carry oxygen at a lower blood pressure than red blood cells.

In some embodiments, the oxygen therapeutic is a per luorocarbon (PFC) compound (a PFC or PFC derivative). In some embodiments, the oxygen therapeutic is a PFC compound formulated as an emulsion. Preferably, the PFC compound is an emulsion composed of one or more perfluorochemicals (PFCs), water, salts, and a surfactant, such as Oxygent (Alliance Pharmaceutical Corp., San Diego, CA), a lecithin-stabilized emulsion of a PFC. In some embodiments, the infusion media is a PFC or PFC derivative conjugated to a fluorescent dye. Unlike hemoglobin, Oxygent does not chemically bind the gas molecules, but absorbs and releases them quickly by simple diffusion. Oxygent is therefore more efficient at delivering oxygen to the tissues than hemoglobin. Oxygent is a peril ubron-based emulsion of small particles (about 0.2 micron in diameter, compared to about 7.0 microns for red blood cells). The small size of the particles may enable them to travel around blockages in vessels, or into very distant capillaries. In some embodiments, the infusion media does not comprise an oxygen therapeutic.

Detectable Labels

Examples of detectable substances include, but are not limited to, the following radioisotopes (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹²⁹I, ^{1 1}I, ⁵¹Cr), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors, fluorescent dyes), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkalline phosphatase, acetyl cholinestease), biotinyl groups (which can be detected by marked avidin, e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). Optionally, the detectable substance (detectable moiety) is conjugated to an infusion media molecule or they may be a single chemical entity. For example, in some embodiments, the detectable moiety is conjugated to a PFC or PFC derivative.

Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against the infusion media molecule. By way of example, if the antibody having specificity against an infusion media molecule is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labeled with a detectable substance.

Methods for conjugating or labeling the antibodies discussed above may be readily accomplished by one of ordinary skill in the art. (See, for example, imman. Methods In Enzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B. Jakoby and Wiehek (eds.), Academic Press, New York, p. 30, 1974; and Wilchek and Bayer, "The Avidin-Biotin Complex in Bioanalytical Applications," Anal. Biochem., 1988, 171 : 1 -32, regarding methods for conjugating or labeling the antibodies with an enzyme or ligand binding partner).

Time-resolved fluorometry may be used to detect a signal. For example, the method described in Christopoulos T. . and Diamandis E.P., Anal. Chem., 1992:64:342-346 may be used with a conventional time-resolved fluorometer.

Therefore, in accordance with an embodiment of the invention, a method is provided wherein an antibody to an infusion media molecule is labeled with an enzyme, a substrate for the enzyme is added wherein the substrate is selected so that the substrate, or a reaction product of the enzyme and substrate, forms fluorescent complexes with a lanthanide metal. A lanthanidc metal is added and the infusion media molecule is quantitated in the sample by measuring fluorescence of the fluorescent complexes. The antibodies specific for the infusion media molecules may be directly or indirectly labeled with an enzyme. Enzymes are selected based on the ability of a substrate of the enzyme, or a reaction product of the enzyme and substrate, to complex with lanthanide metals such as europium and terbium. Examples of suitable enzymes include alkaline phosphatase and beta-galactosidase. Preferably, the enzyme is alkaline phosphatase. The infusion media antibodies may also be indirectly labeled with an enzyme. For example, the antibodies may be conjugated to one partner of a ligand binding pair, and the enzyme may be coupled to the other partner of the ligand binding pair. Representative examples include avidin-biotin. and riboflavin-riboflavin binding protein. Preferably the antibodies are biotinylated, and the enzyme is coupled to streptavidin.

In an embodiment of the method, antibody bound to an infusion media molecule is detected by adding a substrate for the enzyme. The substrate is selected so that in the presence of a lanthanide metal (e.g., europium, terbium, samarium, and dysprosium, preferably europium and terbium), the substrate or a reaction product of the enzyme and substrate, forms a fluorescent complex with the lanthanide metal. Examples of enzymes and substrates for enzymes that provide such fluorescent complexes are described in U.S. Patent No. 5,312,922 to Diamandis. By way of example, when the antibody is directly or indirectly labeled with alkaline phosphatase, the substrate employed in the method may be 4- m ethyl umbeli feryl phosphate, or 5-fluorpsalicyl phosphate. The fluorescence intensity of the complexes can be measured using a time-resolved fluorometer, e.g., a CyberFluor 615 Immoanalyzer (Nordion International, Kanata Ontario).

In some embodiments, the detectable label comprises a fluorescent dye. For example, detectable moieties that may be used include those having absorption and emission wavelengths in the near-infrared (MR) spectrum, between 680 nm and 800 nm. In some embodiments, the detectable moiety is a functional derivative of an infrared dye that is reactive toward free groups on the infusion media molecule, such as free -SI1 groups (e.g., IRDye 800CW Maleimide). In some embodiments, the infusion media is a PFC or PFC derivative conjugated to a fluorescent dye. Examples of fluorescent dyes include but are not limited to IRDye 800CW (Ex_max: 778 nm; Em_max: 794 nm). IRDye 680LT (Ex_max: 680 nm; Em_max: 694 nm), IRDye 750 (Ex ma ^: 66 nm; Em_max: 776 nm), IRDye 700DX (Ex_max: 680 nm; Em_max: 687 nm), IRDye 800RS (Ex_max: 770 nra; Em_max: 786 nm), and IRDye 650 (Ex_max: 651 nm; Em_max: 668 nm) (LI-COR Biotechnology, Lincoln, Nebraska).

In some embodiments in which a conjugate is used for the labeled infusion media, the detectable moiety and/or the infusion media molecule may have functionalities (such as maleimide or other reactive groups) for linking to the other molecule.

Detection Systems

The detection system used to detect and quantify the label in the blood sample obtained from the subject will depend upon the nature of the label in the infusion media. Accordingly, depending upon the detectable substance utilized to label the infusion media, the detection system used to detect the label in the blood sample from the subject may be, for example, a radiation sensor, a Geiger counter, a scintillation counter, an affinity matrix, a plasmon resonance detector, a B1ACORE, a GC detector, an ultraviolet or visible light sensor, an epifluorescence detector, a fluorescence detector (e.g. , fluorescence meter), a fluorescent array, a CCD, a digital imager, a scanner, a con focal imaging device, an optical sensor, a FACS detector, a micro-FACS unit, a temperature sensor, a mass spectrometer, a stereo-specific product detector, an Elisa reagent, an enzyme, an enzyme substrate an antibody, an antigen, a refractive index detector, a polarimeter, a pH detector, a pH-stat device, an ion selective sensor, a calorimeter, a film, a particle counter, an electrochemical sensor, ion/gas selective electrodes, and capillary electrophoresis.

Preferably, in the various embodiments of the invention, the detection system provides an output (i.e., readout or signal) with information concerning the presence, absence, or amount of the label in a blood sample from a subject. For example, the output may be qualitative (e.g., "positive" or "negative"; or "yes" or "no"), or quantitative (e.g., a concentration such as nanograms per milliliter, or other units). The output may be a visual output (e.g., an analog or digital meter (such as a liquid crystal display), or a printout), an audio readout, etc.

Another aspect of the invention is directed to a composition comprising labeled infusion media that may be used in carrying out the methods of the invention, in some embodiments, the labeled infusion media comprises an oxygen therapeutic selected from a perfluorocarbon (PFC) compound or hemoglobin-based oxygen carrier (HBOC). In some embodiments, the label is a radionuclide. In some embodiments, the label is iodine- 129, iodine-131 , or chromium-S i . In some embodiments, the labeled infusion media comprises an emulsion of a PFC, water, salts, and a surfactant, labeled with iodine-131. In some embodiments, the labeled infusion media comprises Hemoglobin Gutamer-250 (bovine) labeled with chromium-51.

Another aspect of the invention comprises an intravascular delivery device, such as a syringe for intravascular injection, containing infusion media and a label, which may be used in carrying out the methods of the invention. Syringes typically include a chamber or reservoir for holding the fluid to be delivered and a plunger to inject the fluid out of the chamber. Syringes of the invention can include a needle or be adapted to accept a needle prior to use.

In some embodiments, the delivery device comprises a chamber containing a composition of the invention in which the infusion media and label are components of the same formulation, e.g., pre-mixed. In some other embodiments, the delivery device comprises a first chamber and a second chamber containing the infusion media and label, respectively, and wherein the first chamber and said the chamber arc separated by a breakable or removable barrier that, upon breaking or removal prior to delivery of the infusion media, establishes fluid communication between the first and second chambers and permits mixing contact between the infusion media and the label.

Intravascular delivery devices of the invention can be prepared using methods known in the art for making such delivery devices. Components of such device devices (e.g., reservoirs, plungers, etc.) typically comprise materials such as glass, metal, and/or plastic, e.g., silicone, polyurethane. polyethylene (PE), polyvinylchloride (PVC), depending upon the desired size of the component, rigidity, bonding capacity, shape memory, sterilization method, and other characteristics.

Another aspect of the invention comprises a kit for blood volume determination, comprising infusion media and a detectable label. The infusion media may be labeled (pre- labeled) or be separate from the label and intended to be mixed prior to use. Optionally, the kit of the invention further comprises printed instructions for carrying its use (e.g., for carrying out the methods of the invention). In some embodiments, the kit of the invention further comprises an intravascular delivery device, such as a syringe, for administering the labeled infusion media to a subject. Optionally, the intravascular delivery device is preloaded with the infusion media (labeled or unlabeled). In some embodiments, the delivery device comprises a chamber containing a composition of the invention in which the infusion media and label are components of the same formulation, e.g., pre-mixed. In some other embodiments, the delivery device comprises a first chamber and a second chamber containing the infusion media and label, respectively, and wherein the first chamber and the second chamber are separated by a breakable or removable barrier that, upon breaking or removal prior to delivery of the infusion media, establishes fluid communication between the first and second chambers and permits mixing contact between the infusion media and the label.

Computer Implementation

The blood determination method of the invention may be computer-implemented. Another aspect of the invention is an article of manufacture useful for determining the blood volume in the vasculature of an individual. The article of the invention comprises computer- executable instructions embodied in a computer-readable medium for performing the blood volume determination method described herein. In some embodiments, the instructions are for performing one or more of the following steps ((a), (b), (c), or (d), or a combination of two or more of foregoing):

(a) introducing a volume of infusion media comprising a pre-dctermined amount of a detectable label (labeled infusion media) into the vascular system of the subject;

(c) obtainin a volume of blood (sample) from the vascular system; and

(d) determining the blood volume in the vascular system based on the amount of label in the volume of blood.

In some embodiments, the step of determining the blood volume in the vascular system of the subject based on the amount of label in the volume of blood obtained from the subject is computer implemented.

In some embodiments, the computer-implemented method further advises a user when sufficient time has passed for the volume of labeled infusion media to equilibrate within the vascular system of the subject, so that a blood sample may then be obtained.

The computer-readable medium may be, for example, a hard disc (hard drive), floppy disc, compact disc (CD), digital video disc (DVD), flash memory device, random access memory (RAM), and read only memory (ROM).

The article of manufacture can include one or more peripheral devices. For example, in some embodiments, the article includes an input device by which a user can input the information used to determine blood volume, and/or patient information such as height, weight, and gender. Preferably, the computer is in operable communication with the detection system for detecting the label in the subject's blood sample.

Optionally, the computer-executable instructions embodied in the computer-readable medium further include instructions for outputting the label signal detected in a subject's blood sample or a calculated blood volume.

In some embodiments, the computer-executable instructions embodied in the computer-readable medium further provide instructions for performing the following steps: (a) recording information obtained from a subject; or (b) outputting information obtained from the subject; or (c) both (a) and (b).

Definitions

As used in this specification, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a sample" includes one sample and more than one such sample. A reference to "an individual" includes a plurality of individuals, and so forth.

The terms "comprising", "consisting of and "consisting essentially of are defined according to their standard meaning. The terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.

The term "ex v vo," as used herein, refers to an environment outside of a subject. Accordingly, a sample of blood collected from a subject is an ex vivo sample of blood as contemplated by the subject invention. In-dwelling embodiments the invention are capable of obtaining samples in vivo.

As used herein, the terms "label" and "tag" refer to substances that may confer a detectable signal, and include, but are not limited to, radiolabels (e.g., radionuclides), enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase, and horseradish peroxidase, ribozyme, a substrate for a replicase such as QB replicase, promoters, dyes, fluorescers, such as fluorescein, isothiocynate, rhodamine compounds, phycocrythrin, phycocyanin, allophycocyanin, o-phth aldehyde, and fluorescamine, chemiluminescers such as isoluminol, sensitizers, coenzymes, enzyme substrates, particles such as latex or carbon particles, liposomes, cells, etc., which may be further labeled with a dye, catalyst or other detectable group. In some embodiments, the label comprises a radiolabel. In some embodiments, the label comprises a fluorescent dye, such as an IR dye having absorption and emission wavelengths in the near-infrared (NIR) spectrum.

The terms "perfl uorocarbon compound'^'' and "PFC compound" are inclusive of fluorocarbons or perfluorocarbons (compounds made up of carbon and fluorine atoms only (such as octal! uoropropane, perfluorohexane, and perfluorodecalin) arranged, for example, in a linear, cyclic, or polycyclic shape) and perfl uorocarbon derivates (perfluorocarbons having some functional group attached).

The terms "subject", "individual", and "patient" are used interchangeably herein to refer to a human or animal. The subject may be male or female. The subject may be any age (adult, child, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a terrestrial mammal such as a dog, cat, or horse. In some embodiments, the subject is a primate such as an ape, chimp, monkey, or human. In some embodiments, the subject is a marine mammal, such as a whale or dolphin. In some embodiments, the subject is a non-mammalian animal, such as a reptile, amphibian, or fish. The methods, compositions, kits, and devices of the invention may be used for assessment of blood volume in the clinical setting, veterinary setting, or preclinical animal research setting, for example.

Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1— Determination of Intravascular Volume of an Anesthetized Animal The objective of the study is the determination of the intravascular volume of an anesthetized animal at the end of an approved surgical training session, just prior to euthanization. Maintenance of appropriate intravascular volume is an important factor in preserving hemodynamic stability (Agarwal R., "Hypervolemia is associated with increased mortality among hemodialysis patients." Hypertension, 2010 Sept: 56(3 ):512-7, Epub 2010 July 12; and Katz SD, "Blood volume assessment in the diagnosis and treatment of chronic heart failure," Am J Med Set, 2007 July, 334( 1 ):47-52). During the minimally invasive surgery training that is part of the approved protocol, preserving cardiovascular function near normal limits in the anesthetized animals is important, and requires ongoing intraoperative hemodynamic stability. This means that being able to assess the vol ume status of the anesthetized animals is of some importance. In order to develop methods for evaluating the intravascular status of the anesthetized animals, the inventors have devised a study to evaluate the efficacy of a perfluorocarbon (Lemal, David M., "Perspective on fluorocarbon chemistry," J. Org. Chem., 2004, Jan., 69 (1 ):1-1 1 ), conjugated with an infrared (IR) dye (Shu, X. et a I., "Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome," Science, 2009 May, 324(5928): 804-807; web site of LI-COR Biotechnology: www.licor.com/bio/products/reagents/irdye/irdye800cw.jsp), in estimating the intravascular volume of anesthetized pigs.

To estimate the animal's blood volume, a modified fluorescent dye dilution technique will be used. A 20cc sample of blood will be drawn from the anesthetized animal, via an existing intravenous line, for the purpose of estimating the background fluorescence using a standard fluorescence meter. Next, a 20cc normal saline sample containing 0.1-0.25 mg of IRDye^® 800CW Infrared Dye (LI-COR Biotechnology, Lincoln, Nebraska), conjugated with 0.1- 0.5gm of perfluorooctyl bromide will be analyzed with a fluorescence meter, and then injected into the anesthetized animal via the existing intravenous (IV) line. Thirty to sixty minutes later, 20cc of the animal's blood will be extracted via the existing IV line, and the fluorescence will be measured with the fluorescence meter. The difference between the background fluorescence and the fluorescence of the second blood sample, of known volume, will be used to estimate the intravascular volume of the anesthetized animal. This protocol will be initiated at the end of the approved surgical training session, and completed just prior to the euthanization of the animal as called for in the original protocol. The entire protocol is expected to require under 60 minutes of elapsed time for each animal. This study will be performed on a maximum of 5 hogs, all of which will be anesthetized under the original protocol.

The fluorescence number of the fluorescent conjugated dye sample will be determined. This will be referred to as X. The background fluorescence number of the initial blood sample will be determined and called O. Some time after the dye sample is injected, a 20cc blood sample will be withdrawn, and the fluorescence number determined. This will be called Y. The net fluorescence number of the second blood sample will be the measured fluorescence number minus the background fluorescence number, i.e., (Y-O). The fluorescent material is assumed to mix freely and uniformly with the blood, such that the concentration in the sample will be the S3.ni 6 BS that in the intravascular space. Specifically, the concentration of the second blood sample ((Y-O)/20cc) should equal the concentration in the blood (X/BV), where BV is the unknown blood volume of interest. Algebraically, the inventors can solve for BV by: BV = (X * 20cc ) / (Y-O).

EXAMPLE 2— Preparation of Fluorescent Dye-Perfluorocarbon Conjugate

The inventors will conjugate an IR dye to a peri uorocarbon to produce a fluorescent dye-perfluorocarbon conjugate. Figure 1 shows the chemical structure of perfubron (perfuoro-octyl bromide), a bromine-substituted fluorocarbon. The bromine atom is unimportant and can be used to link an IR dye. The IR dye to be used (IRDye® 800CW Maleimide; LI-COR Biotechnology, Lincoln, Nebraska) has an emission wavelength of approximately 800 nm; therefore, it will not interfere with red blood emission. IRDye® 800CW Maleimide is a functional derivative of infrared dye IRDye 800CW that is reactive toward free-SH (thiol, sulfydryl) groups. Most molecules that contain free-SH groups can be labeled with maleimide dyes, including IRDye 800 CW Maleimide Infrared Dye. The pcrfluorocarbon to be used (lH,lH2H,2H-Perfluorooctanethiol; CAS: 34451-26-8; Figure 2) has a free sulfhydril (-SH) group which facilitates labeling with maleimide dyes.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

Claims

Claims We claim:

1. A method for measuring the blood volume in a human or animal subject, comprising determining the amount of detectable label in a volume of blood obtained from the subject after a volume of non-blood fluid comprising the detectable label (labeled infusion media) has been introduced and allowed to equilibrate within the subject's vascular system.

2. The method of claim 1, wherein said method comprises:

a. introducing a volume of labeled infusion media into the vascular system of the subject;

b. allowing the introduced volume of labeled infusion media to equilibrate within the vascular system of the subject;

c. obtaining a volume of blood from the vascular system of the subject; and d. determining the blood volume in the vascular system of the subject based on the amount of label in the volume of blood obtained from the subject.

3. The method of claim 1 or 2, wherein the labeled infusion media is a volume expander or oxygen therapeutic.

4. The method of any preceding claim, wherein the labeled infusion media is an oxygen therapeutic selected from a perfluorocarbon (PFC) compound or hemoglobin-based oxygen carrier (HBOC).

5. The method of any preceding claim, wherein the labeled infusion media comprises Hemoglobin Gutamer-250 (bovine).

6. The method of any preceding claim, wherein the labeled infusion media comprises a perfluorocarbon (PFC) or PFC derivative.

7. The method of any of claims 1 -4, wherein the labeled infusion media comprises a perfluorooctylbromide preparation.

8. The method of any of claims 1-4, wherein the labeled infusion media comprises an emulsion of a PFC, water, salts, and a surfactant.

9. The method of claim 8, wherein the emulsion is Oxygent.

10. The method of any preceding claim, wherein the label is a radionuclide.

1 1. The method of claim 9, wherein the label is iodine- 129 or iodine- 13 1.

12. The method of any one of claims 1 -4. wherein the labeled infusion media comprises Oxygent labeled with iodine-131.

13. The method of claim 10, wherein the label is chromium-51.

14. The method of any one of claims 1 -4, wherein the labeled infusion media comprises Hemoglobin Gutamer-250 (bovine) labeled with chromium-51.

15. The method of any preceding claim, wherein the label comprises a radioactive substance and the amount of label in the volume of blood obtained from the subject is detected with a radiation sensor.

16. The method of claim 15, wherein the radiation sensor is a scintillation counter.

17. The method of any one of claims 1 -4, wherein the label is a fluorescent dye.

18. The method of any one of claims 1 -4. wherein the labeled infusion media comprises a perfluorocarbon (PFC) or PFC derivative, and a fluorescent dye.

19. The method of claim 18, wherein the PFC, or PFC derivative, is conjugated to the fluorescent dye.

20. The method of any one f claims 1 -4, wherein the label comprises a fluorescent dye and the amount of label in the volume of blood obtained from the subject is detected with a fluorescence meter.

21 . The method of any preceding claim, wherein the subject is a human.

22. The method of any preceding claim, wherein one or more steps of the method are computer-implemented.

23. A composition comprising labeled infusion media.

24. The composition of claim 18. wherein said labeled infusion media is an oxygen therapeutic selected from a perfluorocarbon (PFC) compound or hemoglobin-based oxygen carrier (HBOC).

25. The composition of claim 23 or 24, wherein said label is a radionuclide.

26. The composition of any preceding claim, wherein said labeled infusion media comprises an emulsion of a PFC, water, salts, and a surfactant, labeled with iodine- 131 .

27. The composition of any one of claims 23 to 25, wherein said labeled infusion media comprises Hemoglobin Gutamcr-250 (bovine) labeled with chromium-51.

28. The composition of claim 23, wherein said label is a fluorescent dye.

29. The composition of claim 23, wherein said labeled infusion media comprises a perfluorocarbon (PFC) or PFC derivative, and a fluorescent dye.

30. The composition of claim 29, wherein the PFC, or PFC derivative, is conjugated to the fluorescent dye.

31. An intravascular delivery device containing infusion media and a label.

32. The device of claim 31 , wherein the device is a syringe for intravascular injection.

33. The device of claim 3 1 or 32, wherein the device comprises a composition of any one of claims 23-30.

34. The device of claim 31 or 32, wherein the device comprises a first chamber and a second chamber containing said infusion media and label, respectively, and wherein said first chamber and said second chamber are separated by a breakable or removable barrier that, upon breaking or removal, established fluid communication between said first and second chambers and permits mixing contact between said infusion media and said label.

35. A kit for blood volume measurement, comprising infusion media and a detectable label.

36. The kit of claim 35, further comprising an intravascular delivery device for administering the infusion media to a subject.

37. The kit of claim 36, wherein said intravascular delivery devices comprises a reservoir, and wherein said infusion media is contained in said reservoir.

38. The kit of any preceding claim, wherein the infusion media is labeled with said detectable label.

39. A computer-readable medium having computer-executable instructions stored thereon for performing the method of any of claims 1 -22.

40. A computer-readable medium having computer-executable instructions stored thereon for performing one or more steps of the method of any of claims 1 -22.