Method of estimating blood volume

Abstract

To provide methods, instruments, and devices for approximation of blood volume in a fluid, such as in a biological fluid collected during a surgical procedure.SOLUTION: The methods and the devices include the use of a RBC flocculant, such as polyDADMAC, and an approximate blood hematocrit for the type of animal, as well as a calculated RBC packing ratio corresponding to the collection device being used. Also provided is a Blood Indicator Panel (BIP), comprising a series of markings calculated from an observed red blood settlement volume, the average animal type hematocrit, and a calculated RBC packing ratio "η" value for the collection device. Pediatric (about 200 ml-250 ml), adult human (about 1,000 ml-1,500 ml), and veterinary (about 500 ml-2,500 ml) collection containers are also disclosed, that include a RBC flocculant, for use in approximating blood volume in a fluid.SELECTED DRAWING: Figure 18

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 445,067, filed Jan. 11, 2017, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] During surgery, blood, saline, and in some cases small tissues are drawn from the subject's body and collected in a collection canister. Typically, such drawing process involves suction. The contents in the collection canister will in many cases contain at least some volume of blood. However, the volume of blood contained is difficult to assess with any degree of accuracy. Although blood loss assessment is very important to the subject's health, it is currently assessed by the attending medical professional / anaesthesiologist only by a crude technique that involves counting saline units employed during the surgical procedure. At the end of the procedure, the anesthesiologist will typically estimate the amount of blood loss by determining the volume of saline used during the entire procedure and subtracting this volume of saline from the total volume of the mixed material collected in the collection canister.

[0003]

[0003] Blood is composed of RBCs, which require a lot of time (ranging from 3 to 6 hours) to settle in the blood / fluid mixture collected during surgery. Apart from the time required to provide a reading of the sinking RBC volume, it has been found that sinking RBC volume does not provide a sufficient approximation of blood volume to assess blood needs. Current techniques for estimating blood loss are fraught with challenges, including human error (counting "used" saline bags, accounting for residual saline), and significant time constraints associated with traditional analysis of blood-containing fluids.

[0004]

[0004] The inability to accurately estimate blood loss during a surgical event can lead to a number of potentially serious health complications for the patient and increased medical costs. For example, underestimating blood loss can lead to failure to provide a subject with a necessary blood transfusion. This can also lead in many cases to the subject becoming anemic, which can require extended hospitalization to stabilize the patient and / or lead to death. Overestimating blood loss can lead to unnecessary transfusions and / or unnecessary medical interventions, and even wasted blood units. This can also lead to an increased risk of disease transmission (e.g., HIV, Hepatitis).

[0005]

[0005] There remains a need in the medical arts for improved techniques and devices for accurately estimating blood loss. Methods are needed to more accurately monitor and estimate blood volume in fluids. There is also a need for fluid collection products suitable for achieving more accurate blood volume estimation, which would serve to reduce medical costs associated with unnecessary medical procedures and resulting interventions. Summary of the Invention [Problem to be solved by the invention]

[0006]

[0006] Generally and in an overall sense, the present invention provides instruments, devices and methods for more reliably and accurately estimating blood volume in liquids, and particularly in liquids collected during surgical procedures. [Means for solving the problem]

[0007]

[0007] In this disclosure, it is to be understood that characterization of red blood cell (RBC) volume separated from plasma-containing fluid, saline, or any other fluid (e.g., urine) does not mean absolute or total separation, but an approximation of steady sedimentation of RBCs in the fluid. RBC sedimentation is evaluated at room temperature and is related to the settling of RBCs in the sample liquid due to gravity (not centrifugation).

[0008]

[0008] A method for estimating blood volume in a liquid. In one aspect, a method is provided for measuring blood loss from a mammal, such as a human or veterinary animal, using an RBC agglutinant. The method, in some embodiments, includes providing a liquid into a container having an RBC agglutinant therein, where RBCs in the blood contained in the liquid will settle, thereby allowing a visual and substantially simultaneous RBC settling volume. Although the settled RBC volume is not equal to the blood volume in the liquid, the RBC settling volume is used to estimate the blood volume in the liquid. The method employs the RBC settling volume observed due to the presence of the RBC agglutinant, the aspect ratio of the collection device, and the average hematocrit to achieve a visually observable estimate of the blood volume in the liquid. Using the present device and method, a visually observable estimate of the blood volume in a blood-containing or potentially blood-containing liquid / fluid can be achieved in less than an hour using the techniques described herein, and can be achieved as quickly as in the range of 15 to 30 minutes. The extremely short time frame facilitated by the presence of the RBC agglutinating agent to achieve RBC sedimentation, and the resulting visual assessment of approximate blood volume, provides the advantageous benefits of maintaining patient health and significantly saving medical personnel.

[0009] The method may also be used to detect the presence of blood in a liquid, and in this manner may be used to test materials (foodstuffs, water, pharmaceuticals, etc.) for the presence of blood contaminants. The material of interest is placed in contact with an RBC agglutinating agent described herein and examined for the presence of RBC sedimentation, which is indicative of the presence of blood in the material.

[0010]

[0010] Blood Indicator Panel. In some aspects, a Blood Indicator Panel (BIP) is provided having predefined sections that are calibrated to correlate to an estimate of blood volume in a fluid containing or suspected of containing blood, and further, a method is provided for preparing a dedicated BIP for blood from a particular mammal (human / non-human animal). A particular collection device associated with the predefined BIP is also presented.

[0011]

[0011] The BIP can be used with any conventional fluid collection device and / or collection bag to provide an estimate of blood volume in a fluid due to the presence of RBC agglutinating agents. The BIP employs a calculation that incorporates the estimated average hematocrit of the blood type being evaluated, as well as the determined device loaded RBC volume for the blood containing fluid being collected. The BIP is specifically provided for veterinary (horse, bovine, canine, feline), human, and human applications (adult, infant). Referencing the BIP provides an efficient approach to estimate blood volume in mixed fluids / liquids by cross-referencing with the sinking RBC volume on the collection device (without having to perform separate calculations). In some embodiments, the BIP can be used without a traditional volumetric scale, having only calibrated blood volume markings to identify the blood volume in the liquid. In such cases, the blood type and device are pre-calibrated to provide an estimated blood volume measurement without reference to sinking RBC levels.

[0012]

[0012] RBC agglutinants and RBC agglutination in fluids / liquids. In one aspect, certain agglutinants are disclosed that are suitable for agglutinating RBCs, thereby promoting sedimentation of the RBCs by gravity. In combination with biological fluids, such as aspirates or other surgical effluents collected during surgery or other medical surgical events, the RBC agglutinants promote rapid settling of the RBCs in the mixed fluid, such as within about 15 to about 30 minutes, compared to the several hours required for RBCs to settle in the fluid in the absence of the RBC agglutinant.

[0013] Agglutination as used in the description of the present invention is defined as the coalescence or formation of clusters of RBCs in a fluid that contains or potentially contains blood. Thus, this method can be used to detect blood contaminants and even to quantify the amount of blood in a material by achieving detection of RBCs and an approximate blood volume in the material according to the method disclosed herein.

[0014] Potentially blood-containing fluids include physiological saline, surgical aspirates, urine, bile, other biological waste, saliva, tissue specimens, digestive fluids, cerebrospinal fluid, lymph, peritoneal fluid, amniotic fluid, and any mixture or combination thereof. In this regard, virtually any component or chemical agent capable of promoting RBC coalescence within less than about 30 minutes at room temperature to provide a stable level of sinking RBCs is considered useful as an RBC agglutinating agent for purposes of providing the device and for use in the present invention. Virtually any component or chemical capable of adding a positive surface charge to negatively charged RBCs is considered useful as part of the device and method to promote more rapid coalescence or aggregation of RBCs in addition to being sufficient to increase the rate of RBC sedimentation.

[0015]

[0015] RBCs present in the blood-containing fluid are bound together by the presence of the RBC agglutinating agent, thus forming heavier particles that sink within the container / bottle / tube / collapsible bag or other vessel in which the sample / material is being collected. As the RBCs sink, the volume of the sinking RBCs is recorded and, taken together with the mammal's (animal / human) given hematocrit and the calculated RBC packing ratio associated with the collection vessel, provides a real-time estimate of the approximate blood volume in the liquid or mixed collected specimen.

[0016] In some embodiments, the agglutinating agent is polydiallyldimethylammonium chloride (polyDADMAC). Virtually any substance or chemical can be used as the RBC agglutinating agent in the practice of the present invention, so long as the substance can promote aggregation of RBCs with each other and can reduce the aggregation time of RBCs and form a stable level of sedimentation, preferably within about 15 minutes. The amount of RBC agglutinating agent should be sufficient to promote stable sedimentation of RBCs present in the fluid within a range of less than about 60 minutes. In some embodiments, the amount of RBC agglutinating agent should be sufficient to promote stable sedimentation of RBCs in the fluid within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes, or within a time range of about 5 minutes to about 20 minutes, at room temperature, without centrifugation. The amount of RBC agglutinating agent should be sufficient to add a cationic charge to the surface of RBCs in the fluid, such as the blood-containing fluid.

[0017] Molecules and substances for use as RBC agglutinants include polymeric and non-polymeric RBC agglutinants. For polymeric RBC agglutinants, the RBC agglutinant must have a molecular weight of at least about 100,000 Da, about 200,000 Da, 300,000 Da, or 400,000 Da, or a mixture thereof, and must have a relatively positive charge. For example, polymeric RBC agglutinants may include PEI, PAM, poly(acylamide-co-acrylate), or any other relatively high molecular weight (about 1,400,000 Da or greater) positively charged polymer that can add a positive charge to the surface of RBCs. Non-polymeric RBC agglutinants may include acids, such as HCl or other acidic molecules.

[0018] Other suitable high molecular weight cationic polymeric RBC flocculants can be prepared by polymerization, such as vinyl addition polymerization of one or more cationic monomers, and copolymerization of one or more cationic monomers with one or more cationic monomers. Although cationic monomeric RBC flocculants can be formed using cationic monomers, it is also possible to react certain nonionic vinyl addition polymers to produce cationic charged polymers. Polymers of this type include those prepared via the reaction of polyacrylamide with dimethylamine and formaldehyde to produce Mannich derivatives.

[0019]

[0019] The RBC flocculant may be used in solid form in aqueous solution, as a water-in-oil emulsion, or dispersed in water. Exemplary cationic polymers that may be employed as RBC flocculants include copolymers or terpolymers of (meth)acrylamide with dimethylaminoethyl methacrylate (DMAEM); dimethylaminoethyl acrylate (DMAEA); diethylaminoethyl acrylate (DEAEA); diethylaminoethyl methacrylate (DEAEM), or their quaternary ammonium forms made with dimethyl sulfate, methyl chloride, or benzyl chloride. In alternative embodiments, the RBC flocculant may include dimethylaminoethyl acrylate methyl chloride quaternary salt-acrylamide copolymer, sodium acrylate-acrylamide copolymer, and hydrolyzed polyacrylamide polymer.

[0020] After the RBCs settle in the collection device or in a collapsible bag with an RBC agglutinant that is designed to follow the dimensions of the collection device, the settled RBC volume can be estimated using graduated markings on the collection device and / or collapsible bag that are calibrated to provide a blood volume estimate. The packing ratio η can be determined. The packing ratio η is calculated using the settled RBC volume relative to the RBC volume after centrifugation. Once the packing ratio is determined, the blood loss volume (V b ), or the total blood volume in the collection fluid in the collection device, is calculated by dividing the settled RBC volume (V m ), the subject's hematocrit (Hct) (or an average HCT determined from multiple similar subjects), and the packing ratio η, can be estimated according to the following formula: V b =V m / (Hct × η) 1)

[0021]

[0021] Typically, in the absence of an aggregating agent, the RBC sedimentation rate or erythrocyte sedimentation rate (ESR) is relatively low or undetectable to the naked eye (see FIG. 18). In the absence of an aggregating agent, it may take hours or days for RBCs to settle in a collection vessel at room temperature without centrifugation. Without intending to be limited to any particular mechanism of action, this may be due, at least in part, to electrostatic repulsion that naturally exists between RBCs, since RBCs are naturally negatively charged. In the presence of an RBC aggregating agent, such as the RBC aggregating agent polyDADMAC, the RBC sedimentation rate is significantly enhanced. In the presence of an RBC aggregating agent, RBC sedimentation occurs in less than about 20 minutes, forming a stable RBC sedimentation volume that is easily visible to the naked eye. The RBC sedimentation volume is considered sufficiently stable for the purposes of the present method when the observable change in the RBC sedimentation volume is less than about 0.5% per minute.

[0022]

[0022] Canisters, collection bags, and containers. Collection devices, including canisters, collection bags, and containers, suitable for collection and visual estimation of blood loss are provided. The collection devices can be in the form of a circle, a square, an octagon, a cylinder, a cone, or virtually any configuration. The collection devices should be sufficiently transparent or opaque to at least allow visual detection of the level of material, such as sedimented RBCs, in the collection device. The collection devices can be devices of solid or flexible materials, such as hard plastic or glass, or plastic materials. The collection devices can have a series of volumetric divisions that are associated with the volume of the device itself and the amount of RBC agglutinant suitable for promoting RBC agglutination. The RBC agglutinant can also be described as having a molecular weight sufficient to allow sedimentation of RBCs associated with the agglutinant in less than 30 minutes, or in a range of less than about 5 minutes, less than about 10 minutes, less than about 15 minutes, or even less than about 20 minutes, at room temperature without centrifugation. The RBC clumping agent may be provided in the collection device as a dry weight clumping agent, may be provided in the collection device as a quantity of clumping agent in a carrier solution, or may be provided as a pre-coating on the collection device (coated uniformly on at least one surface, over the entire surface, over partial areas, or as a strip along the inside of the device, etc.) These descriptions are provided as examples and are not intended to limit the potential embodiments as may be envisioned for the use and practice of the present devices and methods.

[0023] The following list provides exemplary descriptions of the treated and / or flocculant-containing collection vessels that are presented:

[0024]

[0024] A 1,200 ml container (e.g., having a canister configuration), graduated markings of 5 ml and / or 10 ml, one or more inlet ports, and one or more outlet ports (suitable for attachment to a tube suitable for applying a suction force to draw fluid through the tube at the first inlet port and into a receptacle).

[0025]

[0025] A 500 ml container (e.g., having a canister configuration), graduated markings of 1 ml and / or 5 ml, one or more inlet ports, and one or more outlet ports (suitable for attachment to a tube suitable for applying a suction force at the first inlet port to draw biological fluid through the tube and into a receptacle).

[0026]

[0026] A 100 ml, 250 ml or 500 ml container (with a canister or cone configuration), graduated markings of 1 ml and / or 5 ml, one or more inlet ports, one or more outlet ports, where at least one inlet port is adapted to receive a tube for applying suction to draw fluids or other substances into the container. These devices are particularly applicable for pediatric use or smaller surgical procedures (sinus surgery).

[0027]

[0027] A 500 ml collapsible envelope (having graduated markings of 1 ml and / or 5 ml, one or two inlet ports (resealable), and one or more outlet ports suitable for attaching a tube suitable for applying suction to draw fluid through the tube at the first inlet port and into a receptacle).

[0028]

[0028] A collapsible envelope with volumes of 100ml, 200ml, 250ml, 300ml, 400ml, 500ml, 1,200ml, and 2,500ml (optionally with graduated markings of 5ml and / or 10ml), one or two inlet ports (resealable), and one or more outlet ports (suitable for attaching a tube for connecting the bag to a vacuum device for drawing fluid through the tube into a receptacle at the first inlet port) (Figure 13). The collapsible bag should be prepared from a plastic material, as in the case of the saline bag container, and will contain an appropriate amount of flocculant, such as polyDADMAC. The collapsible bag will also have a handle or other loop-like portion (Figure 13, #10) located at one end to facilitate hanging the bag on a fixture such as an iv pole. Additionally, a BIP may be included with the bag or placed as an attachment to the bag, which will provide a visually ascertainable approximate measurement of the volume of blood contained in the collection fluid. The bag may further have conventional volumetric markings, as is common with saline and supply bags.

[0029]

[0029] 100ml, 250ml, and 500ml conical collection receptacles (optionally with graduated markings of 1ml, 5ml, and / or 10ml), RBC agglutinant, one or two inlet ports (resealable), and one or more outlet ports (suitable for attachment of a tube suitable for applying suction to draw fluid through the tube into the receptacle at the first inlet port). These smaller collection devices have particular application for monitoring smaller blood volumes (less than about 500ml) and achieve greater accuracy. These smaller devices, and / or collapsible bags designed to fit into the smaller devices with calibrated blood volume markings specific to the size of the conical receptacle, are particularly useful in neonatal, pediatric, and other critical surgical situations.

[0030] Method for estimating blood volume and blood loss in a fluid. In another aspect, a method for estimating blood volume in a fluid is provided. In a particular embodiment, the method is used to estimate blood volume loss from a patient from fluids collected during a surgical procedure.

[0031]

[0031] The method involves collecting a volume of liquid in a collection device having an RBC agglutinant suitable for applying a relative positive charge to the surface of the RBCs for a sufficient time to allow the RBCs to settle by gravity in the collection device. The volume of settled RBCs after a sufficient time is used together with the calculated RBC packing ratio determined for the particular collection device to calculate an approximate blood volume value in the collected liquid using Equation 1 above, together with the estimated hematocrit value of the animal / human blood type in the fluid. Alternatively, as part of the present invention, these calculations are performed in a calibrated marking panel called a blood indicator panel (BIP), which can be included in the blood collection device. Thus, a quick visual blood volume assessment can be achieved when the RBCs settle in the fluid collected in the collection device containing the agglutinant. In some embodiments, the BIP will have calibration markers that match estimated blood volumes of 50 ml, 100 ml, 200 ml, 400 ml, and 600 ml, as well as volumetric alignment markings for aligning the BIP within a fluid collection device that has conventional volumetric markings (e.g., every 5 ml, 10 ml), such as are common in conventional 1200 ml canisters.

[0032]

[0032] A blood volume assessment system. A fluid collection canister and / or envelope and tubing system. The invention further provides a kit that would have any one or more of the above receptacles with an appropriate amount of RBC agglutinant calibrated with the blood volume section, a first length of tubing suitable for drawing fluid from an area into a first port on the receptacle, and a second length of tubing suitable for connecting to a source of suction and a second port on the receptacle to apply a vacuum into the receptacle. The system may be provided as a kit and may further have an instructional insert. [Brief description of the drawings]

[0033] [Figure 1]

[0033] 50ml of 20%, 40% and 65% bovine blood mixed with 200ul of 3-fold diluted stock solution of Superfloc C-591. 1-Collection canister, 2-Level of sinking RBCs (by gravity), 3-Level of sinking RBCs (by gravity), 4-Level of sinking RBCs (by gravity), 6-Total fluid level in canister, 7-Total level in canister, 10-Coagulant. [Diagram 2]

[0034] FIG. 1 shows containers with various aspect ratios with various erythrocyte sedimentation rates. 1 - graduated conical tube, 2 - graduations with 0.5 ml intervals on the conical tube, 3 - level of sedimented RBCs (gravity), 4 - 100 ml collection canister, 5 - total fluid level in the canister, 6 - level of sedimented RBCs (gravity), 10 - flocculating agent [Figure 3A]

[0035] FIG. 13 is a graph showing the change in sedimentation volume over time (30% blood in saline), the change in RBC sedimentation volume over time for a blood / saline mixture sample having a blood concentration of 30%. [Figure 3B] Sedimentation Volume Change Over Time (40% Blood in Saline) A graph showing the change in erythrocyte sedimentation volume over time for a blood / saline mixture sample having a blood concentration of 40%. [Figure 3C] Sedimentation Volume Change Over Time (65% Blood in Saline) A graph showing the change in RBC sedimentation volume over time for a blood / saline mixture sample having a blood concentration of 65%. [Figure 3D] Experimental setup for measuring average RBC sedimentation volume over time: 1 - glass graduated cylinder, 2 - sedimented RBCs (gravity), 2 - total fluid level. [Figure 4]

[0036] Figure 1. Sedimentation of RBCs in 10ml of 80% blood when diluted with saline to 53.33%, 40%, and 26.7% respectively from left to right. All concentrations sedimented to equal packed RBC volumes. 1- 1200ml collection canister with flocculant, 2-sinking RBC level, 3-total fluid in canister. [Figure 5A]

[0037] Shown is an operating room with a collection canister (8) with agglutinant and cap (15) on an iv pole (6). The cap (15) has an aspiration port (2), an inlet port (4) and a vacuum port (11). The inlet port (4) can be used to add saline or other diluents to the collection canister if needed. The collection canister (8) can have a blood indicator panel (BIP) (5). The aspiration port (2) receives a tube (1) that is used to aspirate biological fluids from the surgical field during a surgical procedure on a patient (7). [Figure 5B] A collection canister (8) is shown having a cap (15) with an aspiration port (2), an inlet port (4) and a vacuum port (11). Fluid aspirated from the patient will be drawn into the collection canister (8) via suction force being pulled through the vacuum port (11) and into the collection canister. [Figure 6]

[0038] Figure 1 shows the flocculant as a particulate suspension (4) can be applied to the interior wall of a container by ultrasonic atomization (3, 4, 5) of the flocculant material in a carrier solution. Any conventional ultrasonic atomization device (2) and syringe pump can be used to coat a collection device such as a 1200 ml canister with the flocculant, for example polyDADMAC. [Figure 7A]

[0039] FIG. 1 shows the application of a polyDADMAC coating (flocculant particles (10)) onto a surface (2) of a collection vessel (1), such as a 1200 ml canister, using an ultrasonic atomization device (3). [Figure 7B]FIG. 1 shows a 1200 ml canister (1) being treated with two batches of polyDADMAC coating on the surface (flocculant particles, 10). [Figure 8]

[0040] FIG. 1 shows a canister to be treated with a coagulant (10) having a cap (1) having a first inlet port (4) connecting the canister by a length of tubing (4) to a container having a volume of a blood mixture, and a second vacuum port (5) connecting the canister to a vacuum source through a length of tubing for the purpose of generating a suction force within the canister. [Figure 9A]

[0041] 1 is a graph showing the change in RBC sinking volume over a 20 minute period for a 20% blood / saline mixture experiment. The total blood / saline mixture volume is 200 ml. Markers are made as part of the BIP to provide an estimate of blood volume (unsinking RBC volume). [Figure 9B] 1 is a graph showing the change in RBC sinking volume over a 20 minute period for a 20% blood / saline mixture experiment. The total blood / saline mixture volume is 400 ml. Markers are made as part of the BIP to provide an estimate of blood volume (unsinking RBC volume). [Figure 9C] 1 is a graph showing the change in RBC sinking volume over a 20 minute period for a 20% blood / saline mixture experiment. The total blood / saline mixture volume is 800 ml. Markers are made as part of the BIP to provide an estimate of blood volume (unsinking RBC volume). [Figure 9D]1 is a graph showing the change in RBC sinking volume over a 20 minute period for a 20% blood / saline mixture experiment. The total blood / saline mixture volume is 1200 ml. Markers are made as part of the BIP to provide an estimate of blood volume (unsinking RBC volume). [Figure 10A]

[0042] 1 is a graph showing the change in RBC sedimentation volume over a 20 minute period for 40% blood / saline, the total blood / saline mixture volume being 200 ml. [Figure 10B] 1 is a graph showing the change in RBC sedimentation volume over a 20 minute period for 40% blood / saline, the total blood / saline mixture volume being 400 ml. [Figure 10C] 1 is a graph showing the change in RBC sedimentation volume over a 20 minute period for 40% blood / saline. The total blood / saline mixture volume is 800 ml. [Figure 10D] Graph showing change in RBC settled volume over a 20 minute time period for 40% blood / saline. Total blood / saline mixture volume is 1200ml. (Note: The volume measurements on each chart are taken from markers prepared for a 1200ml canister treated with agglutinant. The markers are provided as part of a Blood Indicator Panel (BIP) to represent an estimate of blood volume. It is estimated that the markers provide an estimate of blood volume (unsettled RBC volume) which can vary by approximately 20ml.) [Figure 11A]

[0043] Figure 1 shows steady settling of RBCs observed in 10-15 minutes for a 20% bovine blood / saline mixture in a 1200 ml canister (1) coated with polyDADMAC (agglutinant particles, 10). According to the volumetric divisions on the canister, the settling RBC volume in the canister was observed at a volume level of approximately 110 ml (2) and a total volume of 950 ml (3). [Figure 11B] Figure 1 shows steady settling of RBCs after about 15-20 minutes in a 40% bovine blood / saline mixture in a 1200 ml canister (1) coated with polyDADMAC (agglutinant particles, 10). According to conventional volumetric division of the canister, the settling RBC volume in the canister was observed at a volume level of about 240 ml (2) and a total volume of about 1150 ml (3). [Figure 12]

[0044] Three canisters (labeled I, II, and III from left to right) treated with aggregating agent are provided having blood indicator panels (4). As shown, blood indicator panels (4) are presented as separate indicator panels and may be aligned with conventional volumetric markings on the canisters. The blood indicator panels have a series of calibrated markings that provide an approximation of blood volume. The blood / saline mixtures tested in each canister are 50% blood (I), 30% blood (II), and 20% (III). The BIP (see vertical white bar) of canister 12III indicates a settled RBC level of approximately 50ml in the presence of aggregating agent (agglutinating agent particles, 12), which corresponds to a calibrated BIP reading of approximately 116ml. [Figure 13]

[0045] FIG. 1 shows a collapsible plastic bag with coagulant blood receptacle with a BIP (20), an inlet port (3), a vacuum port (4) to be placed on the container, and a second inlet port (15) to allow for the addition of saline or other diluent. The bag should further have a hook (10) adapted to allow for placement of the bag on an IV pole or other attachment. [Figure 14]

[0046] FIG. 1 shows a cone collection device with flocculation agent (critical volume, pediatric use) with a BIP (5), RBC flocculation agent (4), inlet port (2), vacuum port (6) and second inlet port (3). [Figure 15]

[0047] FIG. 1 illustrates a BIP. A typical BIP suitable for use with a 1200 ml collection container may have markers for at least the following calibrated approximate blood volumes: 50 ml, 50 ml, 100 ml, 200 ml, 400 ml, and 600 ml. The BIP will provide an accurate estimate of blood volume in a collection fluid containing up to about 50% less blood. The BIP (20) has a series of calibrated blood volume markings: 6 (50 ml), 7 (100 ml), 8 (200 ml), 9 (400 ml), and 10 (600 ml). The BIP is shown aligned with a volumetric sinking RBC volume reference tool (1), which has conventional volumetric measurements of the collection device, which are 15 (about 25 ml), 4 (about 50 ml), 3 (about 125 ml), and 2 (about 250 ml). The sinking RBC volume does not provide a direct measurement of the volume of blood in the collection fluid (as shown on the BIP), instead the sinking RBC level is a reference point from which an approximate blood volume can be visually ascertained. [Figure 16A]

[0048] Chart data of the rate of RBC sedimentation in control (canisters coated with non-heat treated agglutinant) and experimental (coated with heat treated agglutinant) canisters. Tests were performed using 1000ml of 20% blood saline mixture. The heat treated canister maintained its ability to agglutinate RBCs over a 3 minute period in contact with the agglutinant containing canister with both 20% and 40% blood / saline mixtures introduced into the canister. [Figure 16B] Chart data for the rate of RBC sedimentation in control (canisters coated with non-heat treated agglutinant) and experimental (coated with heat treated agglutinant) canisters. Tests were performed using 500ml of a 40% blood saline mixture. The heat treated canister maintained its ability to agglutinate RBCs over a 6 minute period in contact with the agglutinant containing canister with both 20% and 40% blood / saline mixtures introduced into the canister. [Figure 17A]

[0049] FIG. 1 shows sedimentation of RBCs from bovine blood in canisters coated with high temperature aged flocculant (Panel I (20% blood) compared to a canister of flocculant not treated at high temperature (Panel II (20% blood)). [Figure 17B] FIG. 1 shows sedimentation of RBCs from bovine blood in canisters coated with high temperature aged flocculant (Panel I (40% blood) compared to a canister of flocculant not treated with high temperature (Panel II (40% blood)). [Figure 18]

[0050] FIG. 1 shows a study of horse blood. Comparison of RBC sedimentation in 100 ml containers with and without aggregating agent (volumetric readings estimated from visual inspection using course volumetric markings on the container. RBC sedimentation in 100 ml containers with aggregating agent. 10% blood with aggregating agent (w / F); 20% blood with aggregating agent (w / F); 30% blood with aggregating agent (w / F); 40% blood with aggregating agent (w / F); 50% blood with aggregating agent (w / F); 65% blood with aggregating agent (w / F). 10% blood without aggregating agent (w / o F); 20% blood without aggregating agent (w / o F); 30% blood without aggregating agent (w / o F); 40% blood without aggregating agent (w / o F) (not shown); 50% blood without aggregating agent (w / o F) (not shown ... F) (not shown); in a mixture of 65% blood without aggregating agent (w / o F), no visually detectable sedimentation of RBCs was observed). [Figure 19]

[0051] Figure 1. Study of fresh horse blood. RBC sedimentation from a 61.6% blood / saline mixture in a 1200 ml canister coated with agglutinant. Approximately 400 ml of fresh horse blood was combined with 250 ml of saline. The volume of RBC sedimentation was recorded. RBCs began to sediment within 1 minute (500 ml RBC sedimentation volume). By 15 minutes the RBCs had settled to a volume of approximately 300 ml and remained stable at this sedimentation volume until the end of the observation period. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034]

[0052] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and devices similar or equivalent to those described herein can be used in the practice or testing of the present technology, the preferred methods and devices are described herein.

[0035]

[0053] The term "flocculating agent" as used herein is intended to mean a molecule having a cationic charge that can promote coalescence of RBCs in a fluid at room temperature and further can form a settled RBC mass in less than 30 minutes at room temperature without centrifugation.

[0036]

[0054] Unless the context clearly requires that only a single element is present, referring to an element by the indefinite article "a" or "an" does not exclude the possible presence of more than one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0037]

[0055] "Patient" or "subject" as used herein means an individual having symptoms of or at risk for cancer or other malignancies. A patient may be a human or a non-human animal, and may include animals such as horses, dogs, cows, pigs, or other animals. Similarly, a patient or subject may include a human patient, including an adult or juvenile (e.g., a child). Furthermore, a patient or subject may refer to any organism, preferably a mammal (e.g., a human or a non-human animal), from which it is desired to determine blood volume and / or to determine and / or monitor blood volume by administration of a formulation contemplated herein.

[0038]

[0056] "About" as used herein means within a statistically meaningful range of values, such as a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature, or volume. Such values ​​or ranges may be within an index range, such as typically within 20%, more commonly within 10%, and even more commonly within 5% of a given value or range. The permissible variation encompassed by "about" will depend on the particular system under investigation and can be readily appreciated by one of ordinary skill in the art.

[0039]

[0057] The following examples are presented to demonstrate preferred embodiments of the invention.

[0040] Example 1 - RBC agglutination agent

[0058] This example presents substances that can be used as RBC agglutinating agents in fluids that contain blood or are suspected of containing blood. The following chemicals in Table 1 can be provided as RBC agglutinating agents: [Table 1]

[0041] Laboratory testing and down selection

[0059] All identified chemicals listed in Table 1 were tested and evaluated for suitability. Bovine whole blood purchased from Innovative Research (Novi, Michigan) was used to determine the suitability of each of the above chemicals as an RBC agglutinant. The whole blood contains sodium citrate as an anticoagulant. Saline was used to dilute the whole blood to provide the following blood concentrations: 20%, 30%, 40%, 50%, 65%, and 80%, which were examined in this study.

[0042]

[0060] Gelatin induced RBC clumping but did not cause sedimentation. Finally, gelatin transformed the entire blood / saline mixture sample into a gel structure. Without separating the RBCs from the plasma and saline, gelatin is not suitable for this application.

[0043]

[0061] Dextran 80 + CaCl2 also transformed blood / saline mixture samples into a gel without separating RBCs from plasma and saline.

[0044]

[0062] Acid treatment of blood / saline mixture samples was performed by adding 1.8% of a highly concentrated acid, 6N HCL. The pH of the sample was changed to about 4.5. This acid treatment caused clumping and sedimentation of RBCs. However, using relatively large amounts of strong acid is not cost effective for this application, and strong acid is difficult to handle in the process. Therefore, acid treatment can be used to improve the ESR.

[0045]

[0063] PEI, a positively charged polymer, caused RBC sedimentation from plasma and saline. PEI must be dissolved under acidic conditions (i.e., pH<7.0). In other words, a blood / saline mixture sample at neutral pH requires that acid be added first to lower the pH before PEI can be used to promote RBC sedimentation in biological fluids containing blood. The relative effectiveness of PEI and polyDADMAC was compared. The ESR (erythrocyte sedimentation rate) induced by PEI was found to be lower than that of polyDADMAC. PEI is an acceptable flocculant for RBCs in biological fluids containing blood.

[0046]

[0064] PAMs are polymers that may be either negatively or positively charged. Negatively charged polymers do not induce clumping and sedimentation of negatively charged RBCs. Positively charged PAMs may be expected to be used for this application.

[0047]

[0065] Alum is a positively charged molecule. Alum promoted slow ESR, but at a significantly lower rate and amount. The relative molecular weight of the flocculant was considered for use as a suitable flocculant because it promotes the settling of RBCs by gravity. Studies with PEI and polyDADMAC showed that the higher molecular weight of these polymers caused more rapid RBC settling.

[0048]

[0066] PolyDADMAC is another positively charged polymer. It is also water soluble at neutral pH. PolyDADMAC in the presence of a blood / saline mixture sample has the ability to rapidly induce RBC sedimentation. Only a small amount by weight of polyDADMAC is needed to induce sedimentation. Quantitative studies are described below to determine the fraction of polyDADMAC needed in proportion to the amount of blood in a blood-containing biological mixture. These studies also showed that excessive amounts of polyDADMAC can impede or even stop RBC sedimentation. This may be because the RBCs are coated or surrounded by the positively charged polyDADMAC, which repels each other due to the positive charges rather than the negative charges, thereby impeding and / or preventing aggregation and sedimentation.

[0049]

[0067] Low cost, high molecular weight (at least 100 KDa, 200 KDa, 300 KDa, or about 400 KDa), positively charged polyDADMAC has been identified as a suitable cost-effective flocculant for rapidly inducing RBC sedimentation. Other large molecular weight, positively charged polymeric flocculants (e.g., PEI, PAM, etc.) are also useful in carrying out the method and forming the container vessel. It is also anticipated that acid treatment will be useful as part of the method to achieve RBC sedimentation.

[0050] Example 2 - Estimation of blood loss

[0068] This example demonstrates the use of polyDADMAC material as an effective flocculating agent for RBCs contained in a fluid and the use of this flocculating agent to estimate the total blood volume in fluids containing blood and other liquids, including saline.

[0051]

[0069] High molecular weight (at least 100KDa, 200KDа, 300KDа, or 400KDа) research grade polyDADMAC was obtained from Sigma-Aldrich (catalog numbers 409022 or 409030) along with industrial grade polyDADMAC. The industrial grade product is commercially available from Kemira Chemical under the product name "Superfloc C-591" and contains a concentration of 20% polyDADMAC. This material is used in water treatment. A study was conducted to compare the effectiveness between the research grade product and the industrial grade product. The study found that Superfloc C-591 provided similar RBC sedimentation results as the research grade product. The cost of the industrial grade product was less than the cost of the research grade product. The purity of C-591 varied, which resulted in some differences in RBC aggregation. For the use of research and industrial grade polyDADMAC, the 20 wt.% water stock solution was diluted with water to a 6.67 wt.% working solution to reduce the viscosity of the solution and improve ease of handling. The dry weight of flocculant for the 1200 ml collection canister was 600 mg of polyDADMAC.

[0052]

[0070] Amount of flocculant required to promote RBC sedimentation. Bovine blood and saline mixtures were tested using Superfloc C-591 (as obtained from the manufacturer (viscous liquid, 20 wt.% in H2O) as the RBC flocculant. The amount of flocculant required to promote a more rapid RBC sedimentation rate was examined. It was found that only about 0.4% (v / v%) of working solution of Superfloc C-591 needed to be added to the total blood / saline mixture volume to achieve an acceptable rapidity of RBC sedimentation in the mixture (within 15 minutes). For example, for a 1 liter blood / saline mixture sample, only 4 ml of working solution of Superfloc C-591 was needed, regardless of the blood concentration in the mixture. This translates to about 320 mg of polyDADMAC flocculant for a 1.2 liter canister.

[0053]

[0071] Figure 1 shows three 50 ml volume blood / saline mixture samples with blood concentrations of 20%, 40%, and 65%, respectively (from left to right of the figure). Equal volumes of 200 μl (i.e., 0.4% of 50 ml) of working solution of Superfloc C-591 were added to induce erythrocyte sedimentation at all these blood concentrations. However, as discussed below, it was found that the sedimentation rates differed depending on the concentration of blood in a particular mixture.

[0054]

[0072] Testing has also shown that lower or higher concentrations of working solutions of Superfloc C-591 may be used. For example, concentrations as low as 0.3% (v / v%) and as high as 0.6% (v / v%) have been found to be effective, although precise upper and lower limits have not been determined. Addition of a working solution of Superfloc C-591 at a concentration of 6% (v / v%) to a blood / saline mixture sample having a blood concentration of 20% was found to induce minimal settling, but this may not be the upper limit for settling.

[0055]

[0073] Aspect ratio of the blood container (receptacle or canister). During experimentation, it was found that the RBC settling rate is determined, in part, by the aspect ratio D:H of the container, where D is the diameter of the container and H is the height of the container, for example as shown in FIG. 2. The aspect ratio D:H depends on the shape of the container. When D:H is large, the settling rate is high because a larger D provides a larger area or space for the aggregated RBCs to settle due to gravity. Thus, when different blood collection containers (or canisters) are used, the settling rates will be different.

[0056]

[0074] Another thing that was found during the experiments was that the Superfloc C-591 flocculant flocculates the settled RBCs when it is placed in the container before adding the blood / saline mixture sample, and also when it is added to the container after the container is already filled with the blood / saline mixture sample.

[0057]

[0075] Sedimentation Rate and Blood Concentration. This study also showed that blood concentration in blood / saline mixtures affects RBC sedimentation rate, where the sedimentation rate is higher in samples with low blood concentration versus samples containing high concentration. Figures 3A, 3B, and 3C, for example, show the sedimentation rate of RBCs in 30%, 40%, and 65% blood / saline mixture samples, respectively.

[0058]

[0076] A graduated glass cylinder (Figure 3D) was used in these studies to determine the difference in sedimentation rates. Three 60 ml blood / saline mixture samples with blood concentrations of 30%, 40%, and 65% were used in this experiment. First, a 240 μL (i.e., 0.4% of 60 ml) drop of working solution of Superfloc C-591 was added to the glass cylinder. Then, the 60 ml blood / saline mixture sample was added to the glass cylinder. Separation of RBCs was visually observed by the distinct difference in fluid layers within the glass cylinder, where the sinking RBCs are the more opaque fluid layer toward the bottom of the cylinder and the plasma / saline layer is the clearer fluid layer above the sinking RBC layer. The sinking RBC volume in the cylinder was recorded by referencing the graduated markings on the cylinder, and this was recorded every minute.

[0059]

[0077] At time 0, the sinking RBC volume was set to 60 ml since visual separation had not begun at time 0. After time 0, the RBCs begin to sink from the plasma / saline layer, initially settling toward the bottom of the container at a high sedimentation rate, which decreases over time. In this regard, as an increasing amount of RBCs are separated from the plasma / saline layer, fewer RBCs will separate from the plasma and saline. Eventually, the RBC sedimentation rate becomes so low that the RBC volume change rate is less than 0.5% per minute. At this point, the volume of sinking RBCs is considered stable. In other words, stable sedimentation is achieved when the volume of sinking RBCs changes less than 0.5% per minute. Vm The "packed RBC volume", denoted as , is the volume of the RBC layer at the bottom of the container when the RBC sedimentation is considered to be stable.

[0060]

[0078] FIG. 3A shows that a blood / saline sample mixture with a blood concentration of 30% began RBC sedimentation almost immediately after mixing with the agglutinant. The majority of the sedimentation was completed within 1 minute. Stable RBC sedimentation was achieved approximately 10 minutes after the sample was mixed with the agglutinant. A stable packed RBC volume V m The sedimentation rate was slightly lower for the blood / saline mixture sample with 40% blood concentration (Figure 3B). Most of the RBCs settled in 2 min instead of the 1 min observed for the 30% blood concentration sample. Stable sedimentation for the 40% concentration sample was achieved in about 15 min, and V m The volume of blood in the saline / blood mixture was approximately 14 ml. For samples with 65% blood concentration, sedimentation was slower (Figure 3C). It took more than 60 minutes to observe stable sedimentation. The method for determining blood volume in blood / saline mixtures was optimal and consistent in liquids containing 50% blood or less. Rapid sedimentation is characterized by achieving stable sedimentation of RBCs in saline / blood mixtures within 15 minutes of adding the flocculant at room temperature (without stirring the mixture during sedimentation).

[0061]

[0079] To ensure that the blood concentration in the fluid is maintained at less than about 50%, blood (for optimal RBC separation), additional saline, or other blood diluents may be added. It has been found that rapid sedimentation can still be achieved with blood / saline mixtures with high concentrations of blood. If a slowdown in sedimentation rate is observed, one approach is to add additional volumes of saline to further dilute the blood, which can restart sedimentation. As shown in Figure 4, three 10 ml blood / saline mixture samples with 80% blood concentration (bovine blood containing sodium citrate anticoagulant) were prepared in three bottles. All samples were mixed with 30 μL (about 0.3% of 10 ml) of Superfloc C-591 working solution (about 2 mg dry weight of Superfloc C-591). No sedimentation was observed prior to dilution of the samples. Saline was added to each bottle to dilute the blood concentration. From left to right in Figure 4, 5 ml, 10 ml, and 20 ml of saline were added to dilute the samples to blood concentrations of 53, 3%, 40%, and 26.7%, respectively. The container was swirled slightly to mix the added saline with the sample. Upon dilution, the RBCs began to settle.

[0062]

[0080] The 26.7% and 40% blood concentration mixtures achieved stable sedimentation in approximately 10 minutes. The 53.3% blood concentration mixture achieved stable sedimentation in approximately 16 minutes. As shown in Figure 4, the final packed RBC volume V for all three samples was approximately 1.2 times the volume of saline present. m(i.e., the darker bottom layer at the bottom of the container) are equal. This result was achieved because the initial 10 ml of 80% blood concentration mixture contained an equal amount of 8 ml of blood volume. This investigation showed that: 1) additional saline could be added to dilute the blood / saline mixture to help increase the RBC sedimentation rate, and 2) the total blood volume could be estimated via RBC settling because the volume of RBCs clumped to the bottom of the container (i.e., packed RBC volume) is not affected by the amount of saline in the blood / saline mixture.

[0063]

[0081] It should be noted that further testing can be performed using the testing protocol outlined above with other flocculants or other chemical treatments discussed above to determine optimal flocculant concentrations and blood / saline ratios.

[0064]

[0082] Reproducibility. The RBC sedimentation study was repeated three times using a mixture (Figure 10A) with 40% blood concentration (bovine blood containing sodium citrate anticoagulant). Standard deviation error bars are shown at various points on the line in the plot of Figure 10A. The reproducibility of this experiment was very good, especially at the points on the line when sedimentation approaches stability. Although relatively large variability occurs in the initial state as indicated by the longer standard deviation error bars at the points on the line, these variability are likely due to visual reading errors caused by the rapid volume changes of the settling RBC layer at these points. As sedimentation approaches stability, the variability in the data becomes much smaller. In other words, when sedimentation is stable, the packed RBC volume V m The variation is small.

[0065]

[0083] Estimation of total blood volume. To demonstrate the feasibility of estimating total blood volume, we used the settled RBC volume V mWe developed an algorithm to estimate the total blood volume in a blood / saline mixture based on the actual RBC volume V c is expressed as the total blood volume (determined by centrifugation) and V b and the patient's hematocrit is denoted as Hct, we obtain the following relationship:

number

[0066]

[0084] Hct is available from patient data measured before surgery, and V m Since can be measured based on the above method, V m and V c If the value of the packing ratio (η) between b can be estimated.

[0067]

[0085] The value of the packing ratio η may vary for different blood types (e.g., human vs. non-human animals). It may also be container-specific, since it may vary with the size and shape (aspect ratio) of the container used to collect the blood. In this regard, recall tests have shown that ESR (erythrocyte sedimentation rate) is affected by the shape of the container. Thus, when equal blood types and equal container shapes for blood measurements are used, the packing ratio η maintains equal values.

[0068]

[0086] The compaction ratio η is empirically determined by using a known hematocrit value and a known blood volume in the blood / saline mixture. Preferably, the value of the compaction ratio η will be determined by obtaining an average compaction ratio value from a plurality of blood / saline mixtures having various known blood concentrations. In this regard, by conducting a study using blood from the same animal species (bovine, equine, human) and a container having a predetermined size and shape, a suitable compaction ratio η can be calculated by testing a variety of known blood / saline concentration mixtures with the presence of a coagulant as described herein. Increasing the number of different blood / saline mixtures tested will increase the statistical reliability of the average compaction ratio value obtained. As discussed above, the blood concentration of the collection fluid is about 50% blood or less in some embodiments. In addition, the average hematocrit value can be determined from a plurality of hematocrit values ​​obtained from a typical number of subjects (animal / human). Hematocrit values ​​can be calculated using traditional capillary centrifugation methods as known to those skilled in the art. If the blood volume during a particular collection event is known, the average hematocrit value for a group of animals, such as a group of humans or a group of horses, can be calculated as a value V that can be used in the formulas and methods described herein. c This will provide:

[0069]

[0087] As mentioned above, the value of the packing ratio η is preferably the average packing ratio value calculated from a group of blood / saline mixture samples in a given collection vessel. The actual RBC volume V, facilitated by the presence of the RBC agglutinant, c and sinking RBC volume (usually by gravity, at room temperature) V m is to be determined for multiple individual samples and the average packing ratio value is to be determined. The actual RBC volume V c To determine the hematocrit of the blood added to a mixture sample, the hematocrit is multiplied by the volume of blood added to that sample.

[0070]

[0088] Then, the packed RBC volume V m can be determined for each mixture sample using the conventional volumetric graduated markings on the container as described above. That is, with the presence of flocculant, V can be determined by reading the volume of RBC sedimentation. m can be determined. As part of the method, equal type and amount (concentration in the total fluid mixture volume) of RBC flocculant should be added to the collection vessel. As shown in Table 2, the packing ratio η remained relatively constant even when the concentration of the flocculant, polyDADMAC, was varied. Thus, a range of flocculant concentrations (approximately 0.3%, 0.4%, and 0.75% flocculant in the total liquid volume) can be used to induce RBC sedimentation without significantly affecting the packing ratio. In addition, the data in Table 2 demonstrates that the packing ratio is relatively unaffected by the amount of blood volume in the liquid mixture (blood / saline) when the flocculant concentration remains relatively constant.

[0071]

[0089] The actual RBC volume V of each mixture sample c and the settled packed RBC volume V m Using the above formula, the value of the compaction ratio η for each sample (i.e., each mixture of various blood concentrations) can be determined. In this case, the average compaction ratio value can be calculated. For example, for bovine blood, the calculated average compaction ratio was 1.61 (see Table 2).

[0072]

[0090] To demonstrate the empirical determination of the value of the packing ratio, the value of the packing ratio η was determined for bovine blood purchased from a commercial supplier. These blood materials contained sodium citrate (as an anticoagulant). A graduated plastic canister for volume (ml) was used. This is shown in FIG. 4. The results are shown in Table 2 below. Prior to the experiment, the average hematocrit value Hct of the bovine blood was calculated from multiple bovine blood samples and determined to have an average value of 37.3%. This average Hct is used in the tables below and in the present invention's estimation of blood volume in the samples. Traditional capillary centrifugation methods were used to determine the individual Hct values.

[0073]

[0091] [Table 2]

[0074]

[0092] The resulting packing ratio values ​​from these experiments showed relatively small variations. In these experiments, the blood concentration of all blood / saline mixture samples in the chart above is less than 50%. The reason is that the mixture with a larger amount of blood can be diluted to provide a mixture with less than 50% blood by adding saline. After finding that the Hct is 37.3% and the value of the average packing ratio η is equal to 1.61, the total blood volume of the blood / saline mixture can be estimated using the following formula (obtained from Equation 1):

number

[0075]

[0093] The number 1.67 in the above formula is a calculated value of 1 / 37.3% (average Hct) × 1.61 (average η value from Table 2).

[0076]

[0094] The following table presents a comparison between the actual blood known to be present in each sample compared to the estimated volume (ml) of blood in the sample determined through calculation of blood volume determined using the above formula and the technique described herein.These results demonstrate that the formula and technique provided herein can be used to provide an estimate of blood volume in a liquid sample containing mammalian blood (e.g., bovine blood) in the presence of an RBC agglutinant (e.g., a polymeric agglutinant such as polyDADMAC).In addition, these data show that the estimated blood volume present in the collected mixed blood / saline liquid sample correlates closely with the actual blood volume in the liquid. [Table 3]

[0077]

[0095] The 20% blood described in Table 2 is composed of 200 ml bovine blood and 800 ml saline for every 1000 ml of fluid mixture.

[0078] Example 3 - Fluid Collection Canister

[0096] This example demonstrates the preparation of a particular fluid collection container with an RBC agglutinating agent.

[0079]

[0097] Blood-Containing Liquid Collection Container. The fluid collection container used in the following examples is a 1200 ml suction canister as shown in Figure 7B. This canister (1) was coated with a flocculant, which is polyDADMAC. This flocculant appears as a thin layer of dispersed flocculant particles (2) on the bottom and walls of the canister. Flocculant - PolyDADMAC. Kemira's "Superfloc C-591" was used as the source of polyDADMAC. The purity quality of this product was inconsistent. Therefore, the Sigma Aldrich version of high molecular weight (200-350 KDa) 20% polyDADMAC (Sigma catalog #409022) was used. Results showed that the Sigma version of polyDADMAC significantly improved the test results. However, it is anticipated that virtually any number of different sources of flocculants may be used in the practice of the present invention and in the creation of treated fluid collection vessels containing the flocculants described herein.

[0080] Example 4 - Optimization of PolyDADMAC as a Flocculant for 1200 ml Collection Canisters for Estimating Blood Volume of Blood-Containing Mixtures

[0098] In the operating room, a biological fluid waste collection canister may be used to collect a volume of fluid that will include an unknown volume of blood. The volume of blood in a 1200 ml canister volume may vary from 10 ml to 1200 ml. Also, the volume of blood in the collected fluid may vary depending on the specific species of animal, sex, and weight of the patient, and even depending on the particular medical procedure being performed. However, the volume of blood in the collected fluid in the collection canister during a typical adult human surgical procedure generally contains about 20% to about 50% blood. Conventional techniques for blood volume determination used in standard hospital surgical facilities provide only rough estimates of blood volume that are inaccurate by at least 50% to 75% most of the time, and are only available several hours after completing the surgical procedure.

[0081]

[0099] In many cases, the amount of blood in the fluid aspirated during surgery is 50% or less. In those cases where the fluid contains more than 50% blood, the present method and device can be used to accurately determine blood volume by adding saline or other diluents to the fluid to reduce the blood concentration in order to promote sedimentation of RBCs in the fluid due to the presence of the RBC agglutinating agent polyDADMAC.

[0082]

[0100] This example demonstrates that a relatively constant amount of an RBC agglutinating agent such as polyDADMAC can be used to achieve a relatively accurate estimate of blood volume in fluids containing a blood concentration of about 50% or less. This is accomplished by visually reading the volume of sinking RBCs in a calibrated canister containing the RBC agglutinating agent. This volume of sinking RBCs is then used in a calculation to determine the blood volume of the mixture. The value of the sinking RBC volume alone is insufficient to accurately estimate the blood volume in the mixture.

[0083]

[0101] Three different blood / saline mixtures of 40% blood were tested. The volumes of 40% blood / saline tested were 200 ml, 800 ml, and 1200 ml. An aqueous solution of polyDADMAC (20 wt.% in water) from Sigma-Aldrich was diluted with saline to provide a working solution of 6.67 wt.% polyDADMAC in water. This working solution was used to provide an approximate amount of flocculant in this study.

[0084]

[0102] Table 4 presents the optimal amount of aggregating agent in each volume of 40% blood / saline mixture tested. The optimal amount of aggregating agent is shown as the amount of aggregating agent required to achieve the highest rate of red blood cell sedimentation from the mixture. The RBCs of each mixture volume tested achieved a visually discernible level of sedimentation in the canister, where a relatively clear fluid was observed above the visually discernible meniscus of the sinking RBCs approximately 15 minutes after combining the blood / saline mixture with the aggregating agent at room temperature. Sedimentation rates were observed to vary depending on the amount of aggregating agent provided in the mixture.

[0085]

[0103] From Table 4, an average of about 0.75 ml to about 1.5 ml of flocculant working solution (about 50 mg to about 100 mg dry weight polyDADMAC) was optimal to promote rapid RBC sedimentation in a 100 ml volume of blood / saline mixture. For a larger 1,200 ml canister with a volume of about 1200 ml, about 9 ml of flocculant working solution of polyDADMAC (i.e., about 600 mg dry weight polyDADMAC) would be provided at the bottom of the canister or on the walls of the canister for a maximum volume of 1,200 ml of blood / saline mixture, thereby promoting rapid RBC sedimentation. In the following investigations, about 0.75 ml to about 1.5 ml of flocculant working solution was used for every 100 ml (i.e., 0.75% v / v) of blood / saline mixture. This concentration value is slightly higher than the 0.4% and 0.6% used in the study described herein when using the industrial grade flocculant Superflock C-591 version of polyDADMAC.

[0086]

[0104] Based on the indicated polyDADMAC concentration of 0.75% v / v, a 1,200 ml canister capable of collecting up to 1,200 ml blood-containing fluid would require approximately 9 ml of working solution of polyDADMAC (i.e., approximately 600 mg dry weight of polyDADMAC). [Table 4]

[0087]

[0105] Table 4 demonstrates the amount of flocculant that is optimal to achieve RBC sedimentation for various volumes of 40% blood / saline mixture. As shown, 9 ml of the RBC flocculant polyDADMAC (approximately 600 mg dry weight) achieved optimal RBC sedimentation in a volume of 1200 ml blood saline mixture.

[0088] Example 5 - Method of Preparing a Flocculant-Treated Canister

[0106] This example describes various ways in which a flocculant may be provided and distributed in and / or on a fluid collection container, particularly a blood-containing fluid. A particular polymeric flocculant, polyDADMAC, is used in this example, although many other polymeric or non-polymeric flocculants may be used in the practice of the invention to provide the methods and devices described herein.

[0089]

[0107] Vertical band coating on canister wall. 1,200 canisters were coated with 9 ml of material containing 600 mg of agglutinant. The canisters were coated in the form of a 1" wide vertical band within the canister. In this way, only the agglutinant that is soaked up by the blood / saline mixture is dissolved or released into the mixture, thereby causing RBC sedimentation. In other words, the vertical band of polyDADMAC coating can achieve a controlled release of agglutinant that is proportional to the volume of blood / saline mixture.

[0090]

[0108] Two methods were used to apply the vertical band coating. One method was to use a brush to provide a 1" band directly on the canister wall. The other method was to apply the flocculant coating onto a 1" wide clear tape and adhere the clear tape to the canister wall. Because the canister has a hydrophobic surface, it can be difficult to get the flocculant solution to stay on the wall. Ozone treatment was developed to turn the hydrophobic canister walls into hydrophilic ones. This technique was used to improve flocculant adhesion.

[0091]

[0109] After coating with a vertical band of flocculant, the canisters were tested with bovine blood purchased from a commercial supplier. The canisters with the band of flocculant promoted RBC sedimentation; however, the sedimentation rate was somewhat slow. It took more than 20 minutes for the RBCs to settle to the bottom of the canister. In general, a higher RBC sedimentation rate is desirable. Without intending to be limited to any theory or specific mechanism of operation, the slower rate may be related, at least in part, to the time it takes for the flocculant coating to dissolve and spread throughout the blood / saline volume. It takes longer for the flocculant to spread from a single band coating throughout the entire blood / saline mixture. A thinner coating distributed throughout the canister is expected to accelerate the dissolution and distribution of the flocculant.

[0092]

[0110] Uniform Coating of the Entire Canister Using Ultrasonic Atomization Technology. An ultrasonic-based method was used to coat the inside of the blood collection canister. Ultrasonic atomization coating is not spray coating per se, but rather low-velocity (usually 3-5 inches per second) coating at atmospheric pressure. A piezoelectric transducer converts electrical input into mechanical energy in the form of vibrations. High-frequency sound vibrations atomize the liquid into a fine mist spray (Figure 6). The low-velocity spray without pressure greatly reduces the amount of overspray because the droplets have a tendency to settle on the substrate rather than bouncing off it. The mist spray pattern can be precisely controlled and shaped. Using these types of specialized spray shaping equipment, spray patterns as small as 0.070 inches wide to as large as 1-2 feet wide can be generated. The atomization device used in this process has a 60 kHz ultrasonic nozzle.

[0093]

[0111] Figure 7 shows the manual coating process of a canister using an ultrasonic sprayer. The tip of the sprayer moves along the canister wall, starting from the bottom of the canister and moving upwards in stages to the top of the canister. The manual coating process is guided so that the area coated by the misty spray changes from a clear wall to a cloudy surface, thereby covering the entire canister wall, and making the coating as uniform as possible based on visual observation. In the future, an automatic coating process may be developed to achieve a uniform coating.

[0094]

[0112] Multiple parameters were tested by using various concentrations of polyDADMAC and incorporating highly vaporized alcohols such as methanol and ethanol into the spray solution to accelerate the drying process. A working solution of polyDADMAC (prepared in DI water) provided an optimal misty spray for depositing the flocculant using an ultrasonic atomization device at an intensity setting of "10" (Figure 7A). The syringe pump setting was 60 ml / h. Approximately 4.5 ml of flocculant coating solution was required to cover the entire 1,200 ml canister wall. Thus, two batches of flocculant coating were required to apply 9 ml of flocculant solution onto the canister. A 9 ml volume of flocculant solution contains approximately 600 mg of polyDADMAC.

[0095]

[0113] The canister was allowed to dry completely between each coat for several hours inside an oven or for 24 hours at room temperature. Figure 7B shows a canister with two batches of coatings applied across the entire wall of the canister. It is possible to increase the concentration of the coating solution by using a more powerful ultrasonic atomization device. In this manner, a single batch of coating can be used to provide the aggregate material concentrations described above, providing all of the polyDADMAC contained in 4.5 ml of solution rather than a 9 ml volume.

[0096]

[0114] The following protocol provides the manual steps for preparing a canister to be treated (coated) with a flocculant.

[0097]

[0115] Preparing the coating 1. Prepare PolyDADMAC working solution by mixing 1 part of 20 wt.% PolyDADMAC (Sigma catalog #409022) with 2 parts DI water. 2. Fill a 60 ml syringe with PolyDADMAC working solution. 3. Set the ultrasonic nebulization device to an intensity setting of "10." 4. Set the syringe pump speed to 60 ml / hr to deliver a volume of 4.5 ml.

[0098]

[0116] Manual Coating Process 1. Turn on the ultrasonic atomization device and start moving the ultrasonic nozzle from the bottom of the canister, applying the misty spray to the walls and bottom of the canister. 2. Move the ultrasonic nozzle stepwise from the bottom to the top of the canister along a spiral path, making sure that every spot on the wall is coated with PolyDADMAC. The areas coated with PolyDADMAC mist have a misty appearance as seen in the photo below. 3. Usually 4.5ml of diluted PolyDADMAC is sufficient for one coating of an entire canister. 4. Allow the coated canister to dry overnight at room temperature. 5. After the first layer of coating has dried, a second layer is applied to the canister in a manner similar to that described above. After two layers of coating, a total of 9 ml of diluted PolyDADMAC will be applied to the canister. 6. Again, allow the canister to dry overnight. The canister is now ready to use.

[0099] Example 6 - Estimation of the packing ratio (η) of 1200 ml canisters coated with PolyDADMAC

[0117] A 1200 ml canister coated with polyDADMAC was prepared using a canister having a volume of 1,200 ml and having the dimensions of a biological waste canister employed in an operating theatre. A typical configuration of such a canister in an operating theatre set-up is provided in Figure 5. First, the packing ratio η associated with this coated canister was determined.

[0100]

[0118] The study used bovine blood purchased from a commercial supplier, which was kept refrigerated and warmed to 23°C at the time of testing, and contained sodium citrate to prevent clotting.

[0101]

[0119] Bovine blood and saline were added to the flocculant-treated canister to achieve a predetermined blood / saline ratio. To simulate the process of providing blood-containing liquid into a collection canister during steady-state operation, the flocculant-treated canister was provided with, for example, a 600 ml volume of blood / saline mixture (40% blood) according to the following procedure: Canister A was prepared to have 240 ml of blood and canister B was prepared to contain 360 ml of saline. A serological pipette was used as the aspirating probe. The aspirating probe was connected to an inlet port (patient port) on the canister via tubing. Additionally, a second port on the canister was used to connect the canister to a vacuum line, and the vacuum line was used to apply a vacuum into the canister. Under vacuum, the pipette was placed into a container with blood or a container with saline to alternatively aspirate the respective fluid into the flocculant-containing canister (polyDADMAC) (FIG. 8). This provided 40% blood for a total fluid volume of 600 ml in the canister.

[0102]

[0120] After all the blood and saline were aspirated into the third canister, the mixture was monitored to assess the separation / settling of RBCs away from the plasma and saline in the presence of the agglutinant. The RBC sink volume line was recorded every minute for 20 minutes.

[0103]

[0121] Studies were conducted using various volumes of 40% blood / saline mixture solutions (200 ml, 400 ml, 800 ml, and 1200 ml). Each study was repeated three times. Figures 10A-10D show the change in RBC sedimentation volume over 20 minutes using a 40% blood mixture. Studies were also conducted three times using 20% ​​blood / saline mixture solutions with volumes of 200 ml, 400 ml, 800 ml, and 1200 ml (Figures 9A-9D). Two blood concentrations, 20% and 40%, and four volumes of these blood concentrations from 200 ml to 1200 ml were examined, with the aim of obtaining an average packing ratio η that would be valid for various volumes of fluid collected in the canister and various blood concentration mixtures.

[0104]

[0122] For a 20% blood mixture (20% blood / 80% saline), RBCs settled very quickly in the canisters treated with the flocculant. At room temperature, 30 ml (V m ) reached visually observable subsidence of RBCs (Figure 11A). The average V from triplicate experiments for all four different total mixture volumes of 20% and 40% blood preparations. m and the calculated V ic (Based on the measured hematocrit of bovine blood (35.4%) and the unknown blood volume used in this study) are listed in Table 5.

[0105]

[0123] These experiments demonstrated that the polyDADMAC coating promoted rapid sedimentation of RBCs from blood / saline mixtures within 15 minutes at room temperature. In the absence of a flocculant, sedimentation of RBCs in these blood / saline mixtures at room temperature would require 3-6 hours.

[0106]

[0124] Estimation of the packing ratio η. Using the data from the above experiment, we estimate the sinking RBC volume (V m ) and actual RBC volume (V c) can be empirically determined. Table 5 shows empirically determined packing ratios for a 1200 ml Medi-Vac canister with an average value of 1.20. Because this packing ratio encompasses a wide range of volumes from 200 ml to 1200 ml as well as a wide range of blood concentrations, the variability in the packing ratio is relatively large, exceeding 13%. These variabilities in the packing ratio also affect the variability in the blood volume loss estimates. The following values ​​of packing ratio η were calculated for each of the individual blood mixtures: The average value η was then calculated: [Table 5]

[0107]

[0125] In this study, the average hematocrit of the bovine blood used was 35.4%, resulting in an average packing ratio η of 1.2. Using the formula in Equation 1, the following formula was developed to estimate the volume of blood in the fluid collected in the flocculant-containing canister:

number

[0108]

[0126] Blood loss estimates for each blood mixture 1-8 are presented below in Table 6 and compared to the known blood volume in the sample. [Table 6]

[0109]

[0127] As demonstrated in the table above, the amount of blood loss calculated using this formula correlated with the actual blood volume in the fluid. Thus, the method and device demonstrated to provide a contemporaneous visual indicator tool for blood volume loss for physicians / healthcare professionals in the surgical setting, which is more accurate than traditional approaches (evaluation using saline bags and / or post-operative estimation from the totality of the patient's fluid collection).

[0110]

[0128] Using the above formula, a blood indicator panel was devised based on the observed volume (ml) of settled RBCs in a graduated canister in the presence of aggregating agents without the need for any electrical, temperature, or other instrumentation procedures, which provides a direct visual tool for estimating the total blood volume in the collection fluid.

[0111] Example 7 - Creation of a Blood Volume Indicator Panel for Assessment of Blood Volume in Biological Fluid Receptacles

[0129] To allow a user to easily estimate blood volume via visual inspection of the sinking RBCs in a receptacle (such as a flocculant-containing canister), a blood volume indicator panel having calibrated markings is provided for the fluid collection receptacle, which indicates an approximation of the total blood volume in the collection fluid from the level of sinking RBCs in the presence of flocculant in the collection receptacle. It is contemplated that these collection receptacles may or may not include conventional volumetric scales.

[0112]

[0130] To create the graduated markings for the present receptacle (canister, etc.) blood volume indicator panel, the following formula is used: V m =V b ×Hct×η2)

[0113]

[0131] Equation 2 takes the average Hct calculated from multiple animals / humans of similar age, sex, and species. For example, for adult human males, the average Hct is approximately 45%.

[0114]

[0132] In this study, a blood indicator panel for large mammals was created using the following formulas:

[0115]

[0133] formula 3 V m =0.43×V b

[0116]

[0134] This formula, when calculated for bovine blood (Table 5), uses an average Hct for bovine blood of 35.4% and an average packing ratio η=1.20.

[0117]

[0135] A 50 ml estimated blood volume mark is provided on the blood volume indicator panel, which corresponds to a visually discernible settled RBC volume of approximately 21.5 ml (see Table 7) using Equation 3. A 100 ml estimated blood volume calibration mark may be generated on the blood volume indicator panel, which corresponds to the 43.02 ml settled RBC volume line of the receptacle, and so on.

[0118]

[0136] The graduated markings of the blood volume indicator panel provide a visually recognizable series of markings that are not related to measuring the volume of material in the canister, but rather to approximating the blood volume in the collected fluid in the canister. An illustration of a typical blood indicator panel (BIP) is provided in FIG. 15 (see panel 6 on the left, with calibrated markings of 50 ml, 100 ml, 200 ml, 400 ml, 600 ml). Having a blood indicator panel on a conventional collection vessel with standard volumetric markings (see FIG. 16, panel 1 on the right) would provide a direct visual estimate of the blood volume in the collected fluid without the need to perform mathematical calculations or other manipulation of the collected or settled material. As shown, the blood volume indicated by the calibrated markings of the BPI does not match the conventional volume of the sinking RBCs in the fluid. Instead, the volume of the sinking RBCs is used as part of a calculation along with the hematocrit and a given aspect ratio to provide an estimate of the blood volume. In the absence of aggregating agents, it is not possible to estimate the blood volume in the liquid within a range less than about 3-6 hours, because, among other things, sufficient settling of RBCs has not yet begun at that time. In addition, the presence of aggregating agents only promotes rapid RBC settling, but does not provide a rapid estimate of the blood volume in the liquid. As demonstrated in the present results, the volume of sinking RBCs in the liquid was less than about 50% of the actual blood volume found to be contained in the test fluid containing a known blood volume. The volume of sinking RBCs in the presence of aggregating agents must be further corrected to correspond to the average hematocrit and packing ratio of the blood to provide an estimate of the blood volume in the liquid.

[0119]

[0137] BIP panels are created based on the derivation of mean hematocrit (Hct), e.g., based on the mean Hct of humans, cattle, horses, etc. In order to correct for individual patient / animal Hct differences, such as differences in individual Hct due to gender, species, age, etc., the estimated blood volume value shown on the BIP may be adjusted by a factor that corrects for significantly higher or lower individual hematocrit values. For example, if the measured Hct value from a patient is low, e.g., 80% of the typical Hct for an adult human male, the blood volume indicator value on the panel observed for that patient is divided by 80% to provide a closer approximation of the estimated blood volume in the receptacle. More specifically, if the blood volume indicator value on the panel is 50 ml according to the graduated markings on the indicator panel, the actual blood volume in the biological material collected from this patient is calculated to be 62.5 ml, where the patient's Hct is 80% of the typical Hct value. Similarly, if a patient has a high measured Hct, e.g., 110% of a typical adult male's hematocrit, the blood volume indicator on the blood volume indicator panel is divided by 110%, thereby obtaining a lower blood volume. For example, if the blood volume indicator value on the blood volume indicator panel is 100 ml, the actual blood volume loss for that patient is calculated to be 90.9 ml, thereby correcting for a patient with a higher than average Hct (e.g., 10% higher). Figure 8 shows a 1200 ml canister with a blood volume indicator panel.

[0120]

[0138] A particular blood indicator panel is shown in FIG. 12 when placed on a collection canister. This BIP was created to provide blood volume estimation for the volume of a 20% blood / saline mixture or a 40% blood / saline mixture using bovine blood. This blood indicator panel can also be useful for assessing the volume of human blood in a liquid sample, since both bovine and human blood are mammals and blood from bovine and human blood has many characteristics in common, including similar average hematocrit values. [Table 7]

[0121]

[0139] This study successfully developed a prototype BIP using a biological fluid collection canister (1200 ml) coated with the RBC agglutinant polyDADMAC. This size canister is used in operating rooms for adult and pediatric human patients. Evaluation of this prototype using bovine blood demonstrated rapid sedimentation of RBCs in the presence of this exemplary RBC agglutinant and achievement of stable RBC sedimentation within 20 minutes. Specially designed calibration markings on the BIP were designed for this collection canister. These calibration markings can be used to achieve a visual estimate of the total blood volume in the collected mammalian fluid. If the patient's measured hematocrit differs from the typical hematocrit used to make the calibration BIP markings, the calibration blood volumes can be modified to correspond the percentage blood hematocrit to the individual blood volume amounts.

[0122]

[0140] Below we present the average hematocrit values ​​for adult men and women: Normal Hct values: Men - 42 to 52% (mean Hct, 47); Women - 37 to 47% (mean Hct, 42).

[0123] Example 8 - Preparation of 100ml canister with flocculant

[0141] A 100 ml canister was prepared to contain approximately 50 mg of flocculating agent. In this example, the RBC flocculating agent used was polyDADMAC. The flocculating agent was provided as a volume of a polyDADMAC working solution as described herein.

[0124]

[0142] In a typical operating room setting, small volumes of blood-containing fluids and other materials (tissue, urine, non-blood fluids, etc.) will be aspirated from the surgical field. Aspirating these fluids will result in undetermined blood loss from the patient. To accommodate these small, and potentially critical, volumes of blood loss in collected fluids, smaller containers may be provided in accordance with the present invention. Thus, these 100 ml receptacles are provided that contain an RBC agglutinating agent such as polyDADMAC. The 100 ml receptacles are particularly useful for determining blood volumes in small volumes of collected fluid. These devices may be used, for example, in pediatric applications (infants) and in fluid collection procedures where critical volumes are low.

[0125]

[0143] The aspect ratio of this 100 ml collection device was calculated to be 0.96. A small 100 ml vessel with RBC agglutinant was used in the study described in Example 9.

[0126] Example 9 - Estimation of fresh blood loss

[0144] This example is provided to demonstrate the utility of the method and device for use in estimating blood loss in a fluid containing fresh mammalian blood (without anticoagulants). In this example, the non-blood material present in the fluid was saline. This example examines the technique for estimating blood loss using a volume of fresh, unrefrigerated mammalian blood. Also, the blood does not contain calcium citrate or any other anticoagulants. In this study, fresh blood specimens were obtained from adult horses. Thus, the device and method are particularly useful for estimating blood loss in mammals, including humans and veterinary animals (horses, dogs, cats, cows, bulls, sheep, pigs, etc.).

[0127]

[0145] In this example, blood was collected from a live adult horse (approximately 12 years old, weighing 1,200 pounds) with no known clinical pathology and not undergoing any known medication. The animal had been treated for a lame leg and had been given a nerve block to manage pain. Unlike blood collected from commercial suppliers that contain anticoagulants such as sodium citrate (used in the examples above), the blood collected from the horses used in this study was free of anticoagulants or other medications.

[0128]

[0146] A total of 12 canisters with a total volume capacity of 100 ml were used in this study. The canisters were graduated with divisions of 50 ml and 100 ml increments along the side of the canister. The aspect ratio, D:H (diameter to height), of the 100 ml canister was calculated to be about 0.96. For comparison, the aspect ratio of the 1200 ml canister is about 0.61. Typically, as the aspect ratio of the collection device increases, the RBCs contained in any blood in the collection liquid will settle more quickly within the collection device. Therefore, the rate of RBC sedimentation in the 100 ml collection device was predicted to be faster than the rate of RBC sedimentation in the 1200 ml canister under similar conditions in the presence of an RBC flocculant.

[0129]

[0147] A 100 ml dry canister was charged with 50 mg of RBC flocculating agent, polyDADMAC, research grade (Sigma catalog #409022) (see Example 7). Other RBC flocculating agents, including PEI and PAM, as well as industrial grade versions of these flocculating agents, may be expected to be useful in the present method and device.

[0130]

[0148] Fresh horse blood and saline, in the amounts shown in Table 8, were then added to each canister and the RBC sedimentation volume recorded every minute for 20 minutes. [Table 8]

[0131]

[0149] Blood was drawn from an adult horse and a fixed volume of fresh body temperature blood was added to each canister in the amounts indicated above. The lid of each canister was placed in place and the contents mixed to ensure a proper mixture of saline, blood and agglutinating agent. Each canister was left undisturbed at room temperature and observed. The time at which RBC sedimentation was observed was recorded at one minute intervals for up to 30 minutes. Figure 18 shows the RBC sedimentation rates for the various mixtures. Virtually no visually detectable RBC sedimentation was observed in the blood mixtures without the presence of agglutinating agent. In contrast, rapid RBC sedimentation was observed within five minutes at room temperature in all blood mixtures containing agglutinating agent.

[0132]

[0150] Table 9 presents hematocrit values ​​for various large animals. Mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), and packed cell volume (PCV) values ​​can be employed to provide suitable customized blood indicator panels and blood volume estimation methods described in this disclosure. [Table 9] [Table 10] [Table 11]

[0133] Example 10 - Estimation of blood volume in fluid using a canister with fresh horse blood (without anticoagulant) in the presence of a coagulant

[0151] This example was performed by drawing fresh blood from an adult horse (12 years old, approximately 1,200 pounds). The horse's blood does not contain any anticoagulants. The hematocrit of horse blood is between 32% and 53%. The horse's blood was used freshly drawn and was at body temperature (approximately 101° F., 38.3° C.) when combined with saline in the presence of a coagulant, polyDADMAC (600 mg).

[0134]

[0152] A 1,200 ml canister was used that was spray coated with approximately 9 mls of working solution of polyDADMAC as the flocculant, where a total of approximately 600 mg dry weight of polyDADMAC was used to uniformly coat the 1,200 ml canister. Due to the uniform distribution of the flocculant along the walls of the canister, the amount of flocculant that will be released will be proportional to the amount of fluid provided into the canister to be treated.

[0135]

[0153] The volume of sinking RBCs was recorded over time.

[0136]

[0154] A volume of 400 ml of fresh horse blood (not refrigerated, body temperature, no anticoagulant) was placed into the treated canister. A known volume of 250 ml of saline was added to the canister. The total volume of fluid in the canister was 650 ml. This provided a 61.5% blood solution. Immediately after mixing, the volume of settled RBCs, V m (gravity only, no centrifugation) was recorded for up to 30 min.

[0137]

[0155] After 30 minutes of undisturbed settling, the canister was manually agitated and again placed at room temperature. The agitated blood-containing canister was again observed for evidence of RBC settling at room temperature. [Table 12]

[0138]

[0156] After 30 minutes of observation, the canister was manually shaken and allowed to settle. Re-sedimentation of the RBCs occurred and was observed every minute for 30 minutes. The results of the observation of settled RBC levels were as shown in Table 14. [Table 13]

[0139]

[0157] This study demonstrates that the settling volume of RBCs in solutions containing whole fresh blood remains relatively stable for up to 30-40 minutes at room temperature. Upon agitation, the RBCs in the fresh blood sample settle again, producing a discernible RBC settling volume measurement line V m appeared to provide a very rapid response (3-4 min vs. 16-19 min, with a settled RBC volume of approximately 250 ml to 30 ml). The actual known volume of fresh blood present in the fluid was 400 ml.

[0140]

[0158] The average hematocrit of that species (42.5%, average Hct in horses) and V m (observable sinking RBC volume (ml)) and sinking RBC volume (V mUsing a new packing ratio value (n) determined for equine blood through experimentation using multiple evaluations of equine blood samples of Hct information, and a formula using, an overall estimate of blood volume can be calculated, and a visual blood indicator panel can be created and provided along the vertical axis of a collection canister for use with large animals such as horses. This visual blood indicator panel will provide a quickly visually recognizable estimate of blood volume in a biological fluid containing equine blood. The results of developing a blood indicator panel for a processed canister or other receptacle can be used to provide a visual indicator of equine blood volume loss, and in particular, can be used to more accurately assess equine blood loss compared to what is currently available. Here, equine blood indicator panels can be developed employing information and results provided by those skilled in the art of veterinary medicine without the typical prescribed degree of trial-and-error of experimental optimization. Example 11 - Blood loss estimation for use in human pediatric applications

[0159] This example is presented to provide a canister and method for efficient measurement of blood loss in pediatric patients. A pediatric patient as used in this example is defined as an individual up to 12 years of age weighing a maximum of 70 to 80 pounds.

[0141]

[0160] Total blood volume (TBV) in humans is related to body weight. In children, TBV is around 75-80ml / kg, and is higher in the neonatal period (rising to a peak value of 85ml / kg to 105ml / kg by the end of the first month, then gradually decreasing in the following months). Thus, a 3.5kg 2-week-old newborn has a TBV of around 350ml, whereas a 10kg 15-month-old infant has a TBV of around 800ml.

[0142]

[0161] Since the total blood volume of pediatric patients is significantly less, it is particularly important to provide a blood collection and blood loss estimation system and device designed to accurately estimate blood loss from the smaller blood volumes collected from pediatric patients. Accordingly, this specially designed pediatric blood loss estimation device of the present invention is fabricated using a container having agglutinating agents as described herein and a canister section having a total volume capacity of less than 1000 ml, such as about 500 ml (or about 250 ml for infants or neonates).

[0143]

[0162] Since a large and sudden loss of blood volume in a pediatric patient can put the circulation in a dangerous state, blood loss must be carefully monitored to make it possible to detect a blood loss volume of about 12% of the TBV (about 10 ml / kg) in a pediatric patient in particular (assuming that the child is in a stable condition and has a normal blood hemoglobin (Hb) level at the start of the operation).

[0144]

[0163] For example, a suitable pediatric blood loss collection device has a capacity of 250 mls in some embodiments. The canister preferably provides an appropriate aspect ratio D:H for typical pediatric blood loss volumes. The D (diameter) of the device is typically between 2 inches and 3 inches, with an H (height) of about 2 inches to about 3 inches. At these small dimensions, the collected blood loss volume achieves a reasonably rapid, yet monitorable, settling rate of RBCs, thereby alerting the attending physician that the blood loss has reached a volume at which it would be desirable to transfuse the pediatric patient. It is preferable to achieve a settling rate in blood collected in the canister that achieves RBC settling within 15 minutes.

[0145]

[0164] In some embodiments, the 250 ml canister has a conical shape (Figure 14). The flocculating agent may be provided to the container at the time of the surgical intervention event or may be provided to the canister as a pre-treatment (such as by spray coating).

[0146]

[0165] The amount of flocculant to be added to a 250 ml collection device is from about 50 mg to about 150 mg, or about 125 mg, or an amount sufficient to achieve at least 0.3%, 0.4%, or 0.75% of the total volume of the solution.

[0147]

[0166] For illustration purposes, the following average total blood volumes for patients in the pediatric group can be used in the calculations when blood loss of 12% or more occurs: Also, average hematocrits for pediatric patient classes / groups (premature neonates, term neonates, infants) can be calculated and markings of the average hematocrits for these patient groups are provided aligned to one axis of the canister, thereby providing a visual reference for the attending physician or anesthesiologist to refer to and compare with the hematocrits obtained for patients undergoing this procedure. Premature neonates 95ml / kg Full-term neonates: 85ml / kg Infants: 80ml / kg

[0148]

[0167] The mean hematocrit for human children of a particular weight range and / or age, or for adult males or females, and the V m An estimate of total blood volume can be calculated using a formula using the observable sinking RBC volume (ml) and the packing ratio (n) determined for human blood. The sinking RBC volume (V mUsing multiple assessments of human blood samples of V and Hct information, a visual blood volume indicator panel may be provided for humans, particularly pediatric human models, that would be positioned along the vertical axis of the collection canister. The visual blood volume indicator panel would provide a quickly visually recognizable approximation of blood volume contained in a biological fluid containing human blood for volumes less than about 250 ml. Development of a vertically oriented canister or other receptacle with a blood indicator panel for human blood volume assessment in a liquid, and particularly for assessment of small amounts of human blood loss, may be developed by one of skill in the art given the teachings provided herein without more than a typical degree of trial and error.

[0149] Example 12 - Collapsible Processed Container for Collection of Lost Blood

[0168] This example presents a collapsible plastic-like container (bag) that can be used to collect biological fluid loss and to estimate blood loss. Such a collapsible device is believed to be particularly useful in battlefield situations, or in any other situation where space for medical equipment is limited.

[0150]

[0169] The plastic bag container will have a quantity of RBC agglutinant suitable for implementing the RBC sedimentation and blood loss estimation functions described herein. In some embodiments, the bag may be placed within a supporting container, such as a box, canister, or other structure. The bag may further have a number of markings along the vertical axis of the bag corresponding to volumetric measurements (such as millimeters).

[0151]

[0170] In some aspects, a clear plastic bag having a volume capacity of about 1,000 ml containing between about 300 milligrams and about 4,700 milligrams of aggregating agent such as polyDADMAC will be placed into the bag. The bag will have calibrated divisions of 50 ml, 100 ml, 200 ml, 250 ml, 400 ml, 500 ml, 600 ml, 750 ml, and 1 liter markers in some embodiments. The bag can further have a BIP in the form of an adhesive strip or the like that can be placed on the bag and used to provide a visually recognizable indicator of the approximate blood volume in the liquid based on the sinking RBC level in the collection bag / container. An exemplary depiction of this embodiment is presented in FIG. 13. An insert bag is further provided having an RBC aggregating agent and a calibrated BIP for human blood designed for a 1200 ml collection canister, such as the canister shown in FIG. 11A. In such an embodiment, the canister itself does not need to be treated with an RBC agglutination agent; instead, the insert bag would contain the RBC agglutination agent, which may also optionally further have a calibrated BIP for human blood.

[0152] Example 13 - Blood Loss Collection Kit

[0171] Agglutinant-containing canister (1.2 ml, 500 ml, 250 ml, 10 ml) having a blood volume indicator panel may be provided together in a kit with a length of tubing for aspiration and a second length of tubing suitable for adding saline in the canister and / or collapsible envelope.

[0153]

[0172] An instructional insert may be provided in the kit for the end user.

[0154] Example 14 - High temperature aging stability test

[0173] This example demonstrates the stability of the RBC agglutinating agent, polyDADMAC, which maintained activity to achieve RBC coalescence (aggregation) in blood-containing fluids after exposure of polyDADMAC-coated canisters to elevated temperatures.

[0155]

[0174] In this study, polyDADMAC was used as the flocculating agent, provided as a coating on a canister for collecting material, such as biological fluid material collected during surgery that would contain blood components. The coated canisters were incubated at 55°C for 6 weeks (corresponding to a one year storage period at room temperature). The coated canisters were then compared in functional testing after high temperature aging to coated canisters that had not been subjected to high temperature aging testing.

[0156]

[0175] material: 1) Control group - 4 canisters (Cardinal Health) coated with 600 mg of polyDADMAC (Figure 1) and allowed to dry overnight at room temperature (≈22°C). 2) Experimental group - Four canisters (Cardinal Health) were coated with 600 mg of polyDADMAC (Figure 1) and allowed to dry overnight at room temperature (≈22°C). The canisters were then incubated in a convection oven set at 55°C for 6 weeks. 3) Bovine whole blood (Innovative Research Lot #24301) was stored in a refrigerator and allowed to warm to room temperature before the experiment. 4) Room temperature isotonic saline solution (Thermo Scientific, Lot #994448).

[0157]

[0176] method 1) Four canisters were randomly selected from each experimental and control lot. 2) Whole bovine blood (containing sodium citrate) purchased from a commercial supplier was mixed with isotonic saline at room temperature to obtain blood concentrations of 20% and 40% v / v, respectively. The 20% blood mixture had a total volume of 1000 ml and the 40% blood mixture had a total volume of 500 ml. 3) Two tests were performed on the canisters at a time: one experimental canister vs. one control canister. A mixed blood and saline solution was introduced into the canister via vacuum suction. 4) The sedimentation of the red blood cell volume according to the existing divisions on the canister was recorded every minute for 20 minutes. 5) After 20 minutes, images were obtained comparing the two groups. 6) The data was charted as a function of RBC sedimentation volume versus time for comparison.

[0158]

[0177] Results: Figures 16A and 16B compare the settling of RBCs after introducing blood-saline mixtures into control and experimental canisters (heat treated). Tests were performed using 1000 ml of a 20% blood-saline mixture (Figure 16A) and 500 ml of a 40% blood-saline mixture (Figure 16B). Both contained 200 ml of bovine blood. Each test was repeated once. The data show that the volume change curves of RBC settling in the 20% blood test closely overlap. Approximately 15 minutes after introducing the mixture into the control and experimental canisters, all RBC volume settling stabilized at approximately 125 ml. In the 40% blood test, a delay in volume settling occurred in one experiment, but after 15 minutes all volume settling stabilized at approximately 125 ml. Figure 17 shows pictures of the settling RBCs in both the control and experimental canisters. Figure 17A (panels I and II) and Figure 17B (panels I and II) compare the settling of RBCs after introducing a mixture of blood and saline into a control canister and an experimental canister (heat treated). Tests were performed using 1000 ml of a mixture of 20% bovine blood and saline (Figure 17A) and 500 ml of a mixture of 40% blood and saline (Figure 17B). Both fluids were known to contain 200 ml of bovine blood. Each test was repeated once.

[0159]

[0178] The data show that the RBC sedimentation volume change curves for the 20% blood test closely overlap. Approximately 15 minutes after introduction of the mixtures into the control canister (FIG. 17A) and the experimental canister (FIG. 17B), all RBC volume sedimentation levels stabilized at approximately 125 ml.

[0160]

[0179] In the 40% blood test, one experiment experienced a delay in volume sinking, but after 15 minutes the volume of sinking RBCs stabilized at approximately the 125 ml volumetric mark. Figures 17A and 17B show the settled RBC volumes of the control canister (17A) and the experimental canister (17B).

[0161]

[0180] This study demonstrated that there was no discernible difference in functionality between the control canisters and the experimental canisters after heat treatment. The polyDADMAC-coated canisters after a 6-week aging test at 55°C showed no functional degradation of polyDADMAC or a decrease in its effectiveness for promoting the aggregation of RBCs in fluids. The aggregating agent-coated canisters (polyDADMAC-coated canisters) are predicted to have a shelf life of at least one year without loss of aggregating activity to achieve estimated blood volumes in fluids stored at room temperature.

[0162]

[0181] The examples described above are provided to give those of skill in the art a complete disclosure and description of how to make and use embodiments of the methods for predicting selected modifications that may be made to a biomolecule of interest, and are not intended to be limiting to only what is deemed by the inventors to be the scope of the present disclosure. Modifications of the above-described modes for carrying out the present disclosure may be used by those of skill in the art and are intended to be within the scope of the following claims.

[0163]

[0182] It is to be understood that the present disclosure is not limited to particular methods or systems, which can, of course, vary, and that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0164]

[0183] Although several embodiments of the present disclosure have been described, it should be understood that various modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 1. A visual method for estimating blood volume in a fluid obtained from a subject, comprising: receiving a volume of fluid from a subject having a known hematocrit into a container containing an RBC agglutination agent; visually determining the volume of settled RBCs in said container for a time sufficient to allow steady settling of RBCs; determining a volume of blood in the fluid, the level of stable sedimentation of RBCs being calibrated for the RBC packing ratio and average hematocrit of the container to provide a visual approximation of the volume of blood in the fluid; Including, Visual methods.

2. 2. The visual method of claim 1, wherein the RBC agglutination agent is polyDADMAC in an amount of from about 0.3% to about 0.75% in a container having a volume of about 1200 ml.

3. The visual method of claim 1 , wherein the subject is a human.

4. 10. The visual method of claim 1, wherein the subject is a veterinary animal.

5. The volume of blood in a biological fluid is given by the formula V ｍ =V ｂ ×Hct×η The volume V is calculated according to ｂ 2. The visual method of claim 1, wherein:

6. A container comprising at least two inlet ports, an RBC agglutination agent, and a volume capacity of about 100 ml, about 250 ml, about 500 ml, about 1000 ml, about 1,200 ml, or about 2,000 ml.

7. 7. The container of claim 6, wherein the RBC agglutination agent is polyDADMAC in an amount from about 0.3% to about 0.75% and the volume capacity of the container is about 1,200 ml.

8. 7. The container of claim 6, comprising a series of calibrated blood volume markings, said markings not corresponding to volumetric liquid measurements for said container.

9. 7. The container of claim 6, comprising a cap, said cap having at least one inlet port adapted to receive a suction tube and a second port adapted to apply a vacuum suction to said container.

10. A collapsible fluid collection container comprising an RBC agglutinant, a series of calibrated blood volume markings, at least two ports, a loop portion for installation, and a volume capacity of about 250 ml, about 500 ml, about 750 ml, about 1,200 ml, or about 2,500 ml.

11. The collapsible fluid collection container of claim 10 , wherein the RBC agglutination agent comprises polyDADMAC.

12. 1. A blood indicator panel for use in estimating blood volume in a fluid, the panel comprising a series of calibrated markings corresponding to approximate blood volumes in a fluid including mammalian blood.

13. 13. The blood indicator panel of claim 12, wherein the calibrated markings are calculated from a volume measurement of sinking RBCs in the collection device in the presence of an RBC agglutinating agent, a predetermined RBC packing ratio of the fluid collection device, and an average hematocrit value of the subject animal.

14. 14. The blood indicator panel of claim 13, wherein the RBC agglutination agent comprises polyDADMAC.

15. 14. The blood indicator panel of claim 13, wherein the series of calibrated markings does not indicate a metric volumetric measure of liquid in the collection device.

16. 14. The blood indicator panel of claim 13, wherein the series of calibrated markings provide an estimate of blood volume in a fluid material obtained from an adult human being that is collected in the collection device, and the blood indicator panel is calibrated to match an average hematocrit of blood from the adult human being and an RBC packing ratio determined for a collection device having a volume of approximately 1200 ml.

17. 13. The blood indicator panel of claim 12, wherein the animal is a horse, a human, or a cow.

18. 11. The collapsible fluid collection container of claim 10, comprising a bag having a volume of approximately 1200 ml adapted to be placed in a 1200 ml collection canister.

19. 20. The collapsible fluid collection container of claim 18, wherein the bag comprises a blood indicator panel, the blood indicator panel comprising a series of markings calibrated to provide a visual assessment of blood volume in a liquid contained within the bag.

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