Method of estimating blood volume

Abstract

Methods, materials and devices for estimating the amount of blood in a fluid, such as a biological fluid collected during surgery, are disclosed. The methods and devices include the use of an RBC flocculating agent, such as poly-DADMAC, and an approximate blood hematocrit for the animal type, and a calculated RBC fill rate corresponding to the collection device used. A blood indication panel (BIP) is also provided, which includes a series of indicia calculated from the observed erythrocyte sedimentation volume, the average animal type hematocrit, and the calculated RBC fill rate "η" value for the collection device. Pediatric (about 200 ml or 250 ml size containers), adult (about 1000 ml - 1500 ml), and veterinary (about 500 ml - 2500 ml) collection containers containing an RBC flocculating agent are also disclosed for use in estimating the amount of blood in a fluid.

Description

[0001] Related Applications

[0002] This application is a divisional of Chinese national stage application No. 201880006608.1 and claims priority to U.S. Provisional Patent Application No. 62 / 445,067 filed January 11, 2017, which is incorporated herein in its entirety BACKGROUND

[0003] During surgery, blood, saline, and in some cases small tissue, is withdrawn from the subject's body and collected in a collection container. Typically, this withdrawal process is accompanied by suction. In most cases, the components in the collection canister include at least some volume of blood. However, the amount of blood contained is difficult to assess with any precision. Blood loss assessment is critical to the health of the subject, but it is currently assessed only by crude techniques related to the saline unit counts used by the attending physician / anesthetist during the surgical procedure. At the end of the procedure, the anesthetist typically estimates the blood loss by determining the amount of saline used throughout the procedure and then subtracting that saline volume from the total volume of mixed material collected in the collection canister.

[0004] Blood is composed of RBCs, and RBCs require a significant amount of time (more than 3-6 hours) to settle out of the blood / fluid mixture collected at the time of surgery. In addition to the significant amount of time required to provide a settled RBC volume reading, it has been found that the settled RBC volume does not provide a sufficient approximation of the amount of blood for assessing blood requirements. The techniques currently used to estimate blood loss are fraught with challenges, including human error (calculating "used" saline bags from the remaining saline) and strict time constraints associated with routine blood-containing fluid analysis.

[0005] Inaccurate estimation of blood loss during a surgical event results in a number of potentially serious health complications for the patient and increased medical costs. For example, underestimating blood loss can result in a failure to provide the subject with the required transfusion. This in turn results in many cases in the subject becoming anemic, requiring an extended hospital stay to stabilize the patient and / or even death. Overestimating blood loss can result in unnecessary transfusions and / or other unnecessary medical interventions and waste of unit blood. This can also result in an increased risk of disease (e.g., HIV, hepatitis) transmission.

[0006] There remains a need in the medical arts for improved techniques and materials to accurately estimate blood loss. There is a need for methods that more precisely monitor and estimate the amount of blood in a fluid. There is also a need for fluid collection products adapted to provide a more precise approximation of the amount of blood, which will also serve to reduce medical costs associated with unnecessary medical treatments and interventions that occur as a result. SUMMARY

[0007] Overall, the present invention provides materials, apparatus, and methods for more reliable and accurate estimation of fluid volume, particularly blood volume in fluids collected during surgical procedures.

[0008] In this disclosure, it should be understood that the description of the volume of red blood cells (RBCs) separated from a fluid containing plasma, saline, or any other fluid (e.g., urine) does not imply absolute or complete separation, but rather an approximation of the RBCs that have settled stably in the fluid. RBC sedimentation should be assessed at room temperature and involves the sedimentation of RBCs in the sample solution by gravity (without centrifugation).

[0009] Methods for estimating blood volume in fluids. One aspect provides a method for measuring blood loss in mammals (such as humans or veterinary animals) using an RBC flocculant. In some embodiments, the method includes providing a liquid in a container containing an RBC flocculant, wherein RBCs in the blood contained in the liquid will settle to allow for visual estimation of substantially simultaneous RBC settling volumes. While the settled RBC volume is not equal to the blood volume in the liquid, the RBC settling volume can be used to estimate the blood volume in the liquid. The method employs the RBC settling volume observed in the presence of the RBC flocculant, the aspect ratio of the collection device, and the mean hematocrit to provide a visually determinable approximation of the blood volume in the liquid. Using the techniques described herein, the apparatus and method of the present invention can rapidly provide a visually determinable approximation of the blood volume in a liquid / fluid containing or potentially containing blood within less than an hour, or even 15-30 minutes. Calibration to provide a very short timeframe for erythrocyte sedimentation in the presence of an RBC flocculant, and the subsequent visual assessment of approximate blood volume, offers significant advantages for maintaining patient health and saves considerable resources for healthcare professionals.

[0010] This method can also be used to detect the presence of blood in liquids, and in this way, it can be used to test materials (such as food materials, water, pharmaceuticals, etc.) for blood contamination. The material in question is contacted with the RBC flocculant described herein, and the presence of RBC sedimentation is examined. RBC sedimentation indicates the presence of blood in the material.

[0011] Blood indicator panel. In some aspects, a blood indicator panel (BIP) is provided, having a clear demarcation calibrated to correlate with approximate blood volume in a fluid containing or suspected of containing blood, and a method for preparing a blood-specific BIP from a specific mammal (human / non-human). Specific collection devices associated with the defined BIP are also presented.

[0012] BIP can be used in conjunction with any conventional fluid collection device and / or collection bag in the presence of an RBC flocculant to provide an approximation of the blood volume in the fluid. BIP employs a calculation that combines the estimated mean hematocrit of the blood type being evaluated with the determined RBC hematocrit of the device containing the blood fluid. BIP is specifically intended for veterinary animal (horse, cow, dog, cat) and human (adult, infant) applications. Reference BIP provides an efficient way to estimate the blood volume in a mixed fluid / liquid by cross-referencing the volume of RBCs settled on the collection device (without the need for separate calculations). In some embodiments, BIP can be used without conventional volume measurements and includes only a calibrated blood volume marker to identify the blood volume in the liquid. In this case, the blood type and device are pre-calibrated to provide an approximation of the blood volume measurement without the need to reference the settled RBC level.

[0013] RBC flocculants and RBC flocculation in fluids / liquids. In one aspect, specific flocculants are identified that are suitable for flocculating RBCs to facilitate RBC settling by gravity. In combination with biological fluids, such as aspirate or other surgical effluent collected during a surgical procedure or other medical procedure, RBC flocculants will facilitate rapid settling of RBCs in a mixed fluid, such as within about 15 to about 30 minutes, as compared to the hours required for RBC settling in a fluid without an RBC flocculant.

[0014] As used in the description of the present application, flocculation is defined as the coalescence or formation of RBC clusters within a fluid containing blood or potentially containing blood. Thus, the method can be used to detect blood contamination, as well as to quantify the blood volume in a material by providing detection and approximation of the RBCs in the material according to the methods disclosed herein.

[0015] Fluids that can contain blood include physiological saline, surgical aspirate, urine, bile, other biological waste, saliva, tissue preparations, digestive fluids, cerebral fluids, lymphatic fluids, peritoneal fluids, amniotic fluid, and any mixture or combination thereof. In this regard, any moiety or chemical agent that is capable of facilitating RBC coalescence to provide a stable settled RBC level in less than about 30 minutes at room temperature is considered useful as an RBC flocculant to provide the device of the present application and for use in the methods of the present application. It is contemplated that any moiety or chemical entity that can impart a positive surface charge to negatively charged RBCs can be used to facilitate more rapid coalescence or flocculation of RBCs and be sufficient to increase the speed of RBC settling as part of the device and methods of the present application.

[0016] RBCs present in a fluid containing blood associate with each other in the presence of an RBC flocculant and in this manner form a heavier pellet that settles within the collection sample / material container / vial / tube / foldable bag or other container. Once the RBCs settle, the volume of settled RBCs is recorded and used with a predetermined hematocrit of the mammal (animal / human) and a calculated RBC fill rate associated with the collection container to provide a real-time estimate of the approximate blood volume in the liquid or mixed collection sample.

[0017] In some embodiments, the flocculant is polydiallyldimethylammonium chloride (polyDADMAC). Indeed, in the practice of the present invention, any material or chemical can be used as an RBC flocculant so long as the material is capable of promoting RBCs to aggregate with each other, reducing the time of RBC aggregation and forming a stable sedimentation level, preferably within about 15 minutes. The amount of RBC flocculant should be sufficient to promote stable sedimentation of RBCs present in a fluid in less than about 60 minutes. In some embodiments, the amount of RBC flocculant should be an amount sufficient to promote stable sedimentation of RBCs in a fluid at room temperature and in the absence of centrifugation in about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes or about 30 minutes or in the range of about 5 minutes to about 20 minutes. The amount of RBC flocculant should be sufficient to impart a surface cationic charge to RBCs in a liquid, such as a blood containing liquid.

[0018] Molecules and materials used as RBC flocculants include polymeric RBC flocculants and non-polymeric RBC flocculants. For polymeric RBC flocculants, the RBC flocculant should have a molecular weight of at least about 100,000 Da, or about 200,000 Da, 300,000 Da or 400,000 Da, or mixtures of these, and have a relatively positive charge. For example, polymeric RBC flocculants can include PEI, PAM, poly(acrylamide-co-acrylate), or any other relatively high molecular weight (greater than or equal to about 1,400,000 Da) positively charged polymer capable of imparting a positive charge to the surface of RBCs. Non-polymeric RBC flocculants can include acids, such as HC1 or other acid molecules.

[0019] Other suitable high molecular weight cationic polymeric RBC flocculants can be prepared by polymerization, such as by vinyl addition polymerization of one or more cationic monomers and by copolymerization of one or more cationic monomers with one or more non-ionic monomers. While cationic monomers can be used to form cationic polymeric RBC flocculants, certain non-ionic vinyl addition polymers can also be reacted to produce polymers with cationic charges. Such polymers include those prepared by reacting polyacrylamide with dimethylamine and formaldehyde to produce a Mannich derivative.

[0020] RBC flocculants can be used in solid form, in aqueous solution, as a water-in-oil emulsion, or as a dispersion in water. Representative cationic polymers that can be used as RBC flocculants include copolymers and terpolymers of (meth)acrylamide with dimethylaminoethyl methacrylate (DMAEM); dimethylaminoethyl acrylate (DMAEA); diethylaminoethyl acrylate (DEAEA); diethylaminoethyl methacrylate (DEAEM); or their quaternary ammonium forms prepared with dimethyl sulfate, chloromethane, or benzyl chloride. In alternative embodiments, the RBC flocculant may include a dimethylaminoethyl acrylate methyl chloride quaternary ammonium salt-acrylamide copolymer, a sodium acrylate-acrylamide copolymer, and a hydrolyzed polyacrylamide polymer.

[0021] After RBCs have settled in a collection device or a collapsible bag designed to accommodate RBC flocculants, the volume of settled RBCs can be estimated using the calibrated collection device and / or the scale markings on the collapsible bag to provide approximate blood volume. The fill factor η can be determined by comparing the volume of settled RBCs with the volume of RBCs after centrifugation. Once the fill factor is determined, it can be expressed using the volume of settled RBCs (V0) according to the following formula. m Total blood volume, such as blood loss or the total blood volume in the fluid collected in a collection device, is estimated by the hematocrit (Hct) value of the object (or the average HCT determined from many similar objects) and the filling rate η.

[0022] V b =V m / (Hct×η)1)

[0023] Typically, in the absence of flocculants, the RBC sedimentation rate or erythrocyte sedimentation rate (ESR) is relatively low or undetectable to the naked eye (see [link to flocculant]). Figure 18 Without a flocculant, RBCs may settle in the collection container at room temperature without centrifugation, potentially for hours or even days. While not intended to be limited to any particular mechanism, this is likely at least in part due to the naturally occurring electrostatic repulsion between RBCs, as they are inherently negatively charged. In the presence of an RBC flocculant (such as the RBC flocculant polyDADMAC), the RBC settling rate increases significantly. With an RBC flocculant, RBC settling occurs in less than approximately 20 minutes, forming a stable RBC settling volume that is easily visually apparent. An RBC settling volume is considered sufficiently stable for the method of this invention when the observable change in RBC settling volume is less than approximately 0.5% / min.

[0024] Cans, collection bags and containers: Collection devices are provided, including cans, collection bags and containers suitable for collecting and visually estimating the amount of blood loss. The collection devices can take on a circular, square, octagonal, cylindrical, conical or almost any shape. The collection device should be sufficiently transparent or at least not reflective to allow visual detection of the level of material, such as settled RBCs, within the collection device. The collection device can be solid or flexible material (such as hard plastic or glass) or plastic material. The collection device can include a series of volume demarcations related to the volume of the device itself, as well as an amount of RBC flocculating agent suitable for promoting RBC flocculation. The RBC flocculating agent can also be described as having a molecular weight sufficient to allow RBCs to bind to the flocculating agent without the need for centrifugation, to settle at room temperature in less than 30 minutes, or in about 5 minutes, about 10 minutes, about 15 minutes or even about 20 minutes. The RBC flocculating agent can be provided in the collection device as a dry weight of flocculating agent or as an amount of flocculating agent in a carrier solution or as a pre-coat on the collection device (such as uniformly coated on at least one surface, the entire surface, in a partial area or along a strip inside the device). These descriptions are provided as examples and are not intended to provide limitations on the potential embodiments of the intended use and practice of the devices and methods of the present invention.

[0025] The following list shows exemplary descriptions of collection containers containing treatment and / or flocculating agents:

[0026] 1200 ml container (with can configuration), 5 ml and / or 10 ml graduated markings, one or more inlets and one or more outlets (adapted to connect tubes suitable for applying suction to draw fluid into the container through the tube at the first inlet).

[0027] 500 ml container (with can configuration), 1 ml and / or 5 ml graduated markings, one or more inlets and one or more outlets (adapted to connect tubes suitable for applying suction to draw biological fluid into the container through the tube at the first inlet.

[0028] 100 ml, 250 ml or 500 ml container (with can or conical configuration), 1 ml and / or 5 ml graduated markings, one or more inlets, one or more outlets, wherein at least one inlet is adapted to connect a tube for applying suction and drawing fluid and other materials into the container. These devices are particularly suitable for pediatric use as well as smaller surgical procedures (sinus surgery).

[0029] 500 ml collapsible envelope (with 1 ml and / or 5 ml graduated markings, 1 or 2 inlets (re-sealable) and one or more tubes adapted to connect tubes suitable for applying suction to draw fluid into the container through the tube at the first inlet.

[0030] Shrinkable caps (optionally with 5ml and / or 10ml graduations) in volumes of 100ml, 200ml, 250ml, 300ml, 400ml, 500ml, 1200ml, and 2500ml, one or two inlets (resealable) and one or more outlets (suitable for connecting tubing to connect the bag to a vacuum device, thereby drawing fluid into the container through the tubing at the first inlet) Figure 13 The collapsible bag should be made of a plastic material similar to that of a saline bag container and will contain an appropriate amount of flocculant, such as polydADMAC. The collapsible bag will also include a handle or other loop at one end. Figure 13 (#10) to facilitate hanging the bag on an attachment such as an IV pole. A BIP may also be included on the bag or placed as an accessory to the bag, providing a visually identifiable approximate measurement of the amount of blood contained in the collected fluid. The bag may also include standard volume markings, such as those commonly found on saline and infusion bags.

[0031] 100ml, 250ml, and 500ml conical collection containers (optionally with 1ml, 5ml, and / or 10ml graduation marks), RBC flocculant, one or two inlets (reusable), and one or more outlets (suitable for connection to tubing that allows aspiration to draw fluid into the container through the tubing at the first inlet). These smaller collection devices are application-specific and offer greater accuracy for monitoring smaller (less than approximately 500ml) blood volumes. These smaller devices and / or collapsible bags designed to fit within them include calibration blood volume markings specifically for the conical container size, making them particularly suitable for neonatal, pediatric, and other critical surgical situations.

[0032] Methods for estimating blood volume and blood loss in fluids: In another aspect, a method for estimating blood volume in fluids is provided. In a specific embodiment, the method is used to estimate the amount of blood loss in a patient during surgical procedures due to the collection of fluids.

[0033] The method comprises collecting a volume of fluid into a collection device comprising a RBC flocculant adapted to impart a relatively positive charge to the surface of the RBCs for a period of time sufficient to allow the RBCs to settle by gravity within the collection device. The volume of RBCs settled after a sufficient period of time and the estimated hematocrit of the animal / human blood type in the fluid are used in conjunction with a calculated RBC fill rate determined for the particular collection device to calculate an approximate blood volume value in the collected fluid using Equation 1 above. Optionally, these calculations are provided in a calibration marker panel called a Blood Indication Panel (BIP) as part of the present application, which can be included on the blood collection device. Thus, an immediate visual blood volume assessment is provided in the RBCs collected in the sediment of the collection device containing the flocculant. In some embodiments, the BIP will include calibration markers consistent with estimated blood volumes of 50 ml, 100 ml, 200 ml, 400 ml, and 600 ml, as well as volume calibration markers for calibrating the BIP within a fluid collection device of a regular volume marker (e.g. every 5 ml, 10 ml), as is commonly found in regular 1200 ml jars.

[0034] Blood volume assessment system: fluid collection jar and / or envelope and tubing system: The present application also provides a kit which will include any one or more of the above containers, including a suitable amount of RBC flocculant, and calibrated with blood volume demarcations, a first length of tubing adapted to aspirate fluid from a region to a first port on the container, a second length of tubing adapted to connect to an aspiration source and a second port of the container so as to create a vacuum in the container. The system can be provided as a kit and can also include instructions for insertion. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 . 50 ml of 20%, 40% and 65% bovine blood was mixed with 200 μl of 3x diluted Superfloc C-591 stock solution. 1 - collection jar, 2 - RBC sedimentation level (by gravity), 3 - RBC sedimentation level (by gravity), 4 - RBC sedimentation level (by gravity), 6 - total fluid level in jar, 7 - total level in jar, 10 - flocculant.

[0036] Figure 2 . Containers with different aspect ratios have different rates of red blood cell sedimentation. 1 - graduated conical tube, 2 - graduations on conical tube at 0.5 mm intervals, 3 - RBC sedimentation level (gravity), 4 - 100 ml collection jar, 5 - total fluid level in jar, 6 - RBC sedimentation level (gravity), 10 - flocculant.

[0037] Figures 3A-3D . Figure 3A- Sedimentation volume change over time (30% blood in saline). Change in RBC sedimentation volume over time in a sample of blood / saline mixture with a blood concentration of 30%. Figure 3B - Sedimentation volume change over time (40% blood in saline). Change in RBC sedimentation volume over time in a sample of blood / saline mixture with a blood concentration of 40%. Figure 3C - Sedimentation volume change over time (65% blood in saline). Change in RBC sedimentation volume over time in a sample of blood / saline mixture with a blood concentration of 65%. Figure 3D - Setup for experiment to measure average RBC sedimentation volume over time. 1 - glass graduated cylinder, 2 - settled red blood cells (gravity), 2 - total fluid level.

[0038] Figure 4 . Settled RBC in 10 ml of 80% blood from left to right when diluted with saline to 53.33%, 40% and 26.7% respectively. All concentrations set to same RBC hematocrit. 1 - 1200 ml collection canister (with flocculant), 2 - RBC sedimentation level, 3 - total fluid in canister.

[0039] Figures 5A-5B . 5A - Operating room with collection canister (8) and lid (15) with suction port (2), inlet port (4) and vacuum port (11) on intravenous infusion pole (6). Inlet port (4) can be used to add saline or other diluent to the collection canister as needed. Collection canister (8) can include a blood indication panel (BIP) (5). Suction port (2) will house a tube (1) for suctioning biological fluids from the surgical area during surgery on patient (7). 5B - Collection canister (8) will include lid (15) with suction port (2), inlet port (4) and vacuum port (11). Suctioned fluids from the patient will be suctioned into the collection canister (8) through the vacuum port (11).

[0040] Figure 6 . Flocculant can be applied to the interior walls of the container as a particulate suspension (4) 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 collection devices such as a 1200 ml canister containing flocculant such as polyDADMAC.

[0041] Figure 7A . PolyDADMAC coating (flocculant particles (10)) applied to the surface (2) of a collection container (1) such as a 1200 ml canister using an ultrasonic atomization device (3). Figure 7B . Treated 1200 ml canister (1) with two batches of polyDADMAC coating (3) (flocculant particles, 10) on the surface.

[0042] Figure 8 . A canister of flocculant (10) treatment with a lid (1) having a first inlet (4) and a second vacuum port (5), the first inlet (4) connecting the canister to a container of a volume of blood mixture through a length of tubing (4), the second vacuum port (5) connecting the canister to a vacuum source through a length of tubing to create a suction force within the canister.

[0043] Figures 9A-9D . Change in RBC settled volume over a 20 minute period in a 20% blood / saline mixture experiment. Total blood / saline mixture volume was 200ml (1), 400ml (2), 800ml (3), and 1200ml (4) respectively. Markers were formed as part of the Blood Indicators Panel (BIP) to provide an approximation of blood volume (un-settled RBC volume). Figure 9A Figure 9B Figure 9C Figure 9D

[0044] Figures 10A-10D . Change in RBC settled volume over a 20 minute period in a 40% blood / saline mixture. Total blood / saline mixture volume was 200ml (1), 400ml (2), 800ml (3), and 1200ml (4) respectively. Figure 10A-2 Figure 10B-4 Figure 10C-8 Figure 10D-12 (Notes: The volume measurements on each graph are from the markers prepared for a 1200ml canister treated with flocculant. The markers provided as part of the Blood Indicators Panel (BIP) are used to indicate an approximation of blood volume. It is estimated that the markers provide an approximation of blood volume (un-settled RBC volume) that can vary by up to about 20ml.)

[0045] Figure 11A . Stable RBC settling in a 20% bovine blood / saline mixture was observed in a 1200ml canister (1) coated with polyDADMAC (flocculant particles, 10) at 10-15 minutes. According to the volume markings on the canister, the settled RBC volume in the canister was observed to be at a volume level (2) of about 110ml, and the total volume was 950ml (3). Figure 11B . Stable RBC settling in a 40% bovine blood / saline mixture was observed in a 1200ml canister (1) coated with polyDADMAC (flocculant particles, 10) at about 15-20 minutes. According to the regular volume markings on the canister, the settled RBC volume in the canister was observed to be at a volume level (2) of about 240ml, and the total volume was about 1150ml (3).

[0046] Figure 12 ​​​​​​​I, 12 II and 12 III. Three flocculant treated jars (labeled I, II and III from left to right) are prepared to include a blood indicator panel (4). As shown, the blood indicator panel (4) is presented as a stand-alone indicator panel and can be provided with the regular volume markings on the jar. The blood indicator panel includes a series of calibration markings that provide approximate values for blood volume. The blood / saline mixture detected in each jar is 50% blood (I), 30% blood (II) and 20% (III). In jar 12 III, the BIP (see vertical white bar) shows a RBC sedimentation level of about 50 ml in the presence of flocculant (flocculant particles, 12) which corresponds to a reading of about 116 ml of the calibration BIP.

[0047] Figure 13 . Collapsible plastic bag blood container containing flocculant with BIP (20), inlet (3), vacuum port to be placed on the container (4) and a second inlet (15) to allow the addition of saline or other diluent. The bag should also include hooks (10) made to allow the bag to be placed on an IV pole or other mounting.

[0048] Figure 14 . Conical collection device containing flocculant (critical volume and pediatric applications) with BIP (5), RBC flocculant (4), inlet (2), vacuum port (6) and second inlet (3).

[0049] Figure 15 . BIP. A typical BIP for a 1200 ml collection container can include markers for approximate blood volume for at least the following calibrations: 50 ml, 100 ml, 200 ml, 400 ml and 600 ml. The BIP will provide an accurate approximation of the blood volume contained in the collected fluid up to about 50% or less blood. The BIP (20) includes a calibration series of blood volume markers 6 (50 ml), 7 (100 ml), 8 (200 ml), 9 (400 ml) and 10 (600 ml). The BIP is shown with a volume RBC sedimentation volume reference tool (1) which includes regular volume measurements for collection devices 15 (about 25 ml), 4 (about 50 ml), 3 (about 125 ml) and 2 (about 250 ml). The volume of sedimented RBCs does not provide a direct measurement of the blood volume in the collected fluid (indicated on the BIP). Rather, the level of sedimented RBCs is a reference point for an approximate blood volume that can be visually determined.

[0050] Figures 16A-16BGraphical data of RBC sedimentation rate in control (floculant coated cans without heat treatment) and experimental (floculant coated cans with heat treatment). Testing was performed using 1000 ml of 20% blood and saline mixture (16A) and 500 ml of 40% blood and saline mixture (16B). The ability of the heat treated cans to induce RBC flocculation in the 20% and 40% blood / saline mixtures introduced into the cans was measured over 3 minutes ( Figure 16A ) and 6 minutes ( Figure 16B ).

[0051] Figures 17A-17B . Sedimentation of bovine blood RBC in floculant coated cans heat aged compared to floculant cans not heat aged ( Figure 17A , Figure I (20% blood), Figure 17B , Figure II (40% blood)). Figure 17A Figure 17B

[0052] Figure 18 . Horse blood study: RBC sedimentation comparison in 100 ml containers with or without floculant (approximate volume readings were visually inspected using process volume markings on the containers). RBC sedimentation in 100 ml containers with floculant: 10% blood with floculant (w / F); 20% blood with floculant (w / F); 30% blood with floculant (w / F); 40% blood with floculant (w / F); 50% blood with floculant (w / F); 65% blood with floculant (w / F); no visually detectable RBC sedimentation was observed in the following mixtures: 10% blood without floculant (w / o F); 20% blood without floculant (w / o F), 30% blood without floculant (w / o F); 40% blood without floculant (w / o F) (not shown); 50% blood without floculant (w / o F) (not shown); or 65% blood without floculant (w / o F), mixtures were tested at room temperature over the test period (0 to 25 minutes).

[0053] Figure 19 . Horse fresh blood study: RBC sedimentation in a floculant coated 1200 ml can with a 61.6% blood / saline mixture. Approximately 400 ml of fresh horse blood was mixed with 250 ml of saline. The RBC sedimentation volume was recorded. The RBCs began to settle out within 1 minute (500 ml RBC sedimentation volume). The RBCs settled to a volume of approximately 300 ml within 15 minutes and remained stable at that sedimentation volume until the end of the observation period.

[0054] Detailed description of preferred embodiments

[0055] ​​Unless defined otherwise, 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice and testing of the present technology, the preferred methods and materials are described herein.

[0056] The term "flocculating agent" as used herein is intended to mean a molecule that has a cationic charge, is capable of promoting the coalescence of RBCs in a fluid at room temperature, and forms a settled mass of RBCs in less than 30 minutes at room temperature and without centrifugation.

[0057] Reference to elements by the indefinite article "a" or "an" does not exclude the presence of more than one of such element unless the context clearly requires that there be one and only one of the elements. Thus, the indefinite article "a" or "an" is used herein to mean "at least one" or "one or more."

[0058] As used herein, "patient" or "subject" means an individual who has symptoms of a cancer or other malignancy or who is at risk of having a cancer or other malignancy. The patient can be human or non-human and can include, for example, animals such as horses, dogs, cows, pigs, or other animals. Likewise, the patient or subject can include a human patient, including an adult or a juvenile (e.g., a child). Further, the patient or subject can mean any living body, preferably a mammal (e.g., a human or non-human), for which it is desirable to determine and / or monitor the volume of blood from the mammal by administration of a composition contemplated herein.

[0059] As used herein, "about" means within a statistically meaningful range of values, such as a concentration, length, molecular weight, pH, sequence identity, time range, temperature, or volume as described. Such values or ranges can be within an order of magnitude of the given value or range, typically within 20%, more typically within 10%, and even more typically within 5%. The permissible deviation encompassed by "about" will depend on the particular system under study, and can be readily appreciated by one of skill in the art.

[0060] The present application is also directed to the following embodiments:

[0061] Embodiment 1. A visual method for estimating the volume of blood in a fluid obtained from a subject, comprising:

[0062] receiving a volume of fluid from a subject having a known hematocrit into a container comprising an RBC flocculating agent;

[0063] visually determining the volume of settled RBCs in the container for a period of time sufficient to allow the RBCs to settle stably; and

[0064] determining the volume of blood in the fluid, wherein the stable sedimentation level of RBCs is calibrated with the RBC fill rate of the container and the mean corpuscular hematocrit to provide a visual estimate of the volume of blood in the fluid.

[0065] Embodiment 2. The visual method according to embodiment 1, wherein the RBC flocculating agent is polyDADMAC in an amount of about 0.3% to about 0.75% in a container having a volume of about 1200 ml.

[0066] Embodiment 3. The visual method according to embodiment 1, wherein the subject is a human.

[0067] Embodiment 4. The visual method according to embodiment 1, wherein the subject is a veterinary animal.

[0068] Embodiment 5. The visual method according to embodiment 1, wherein the volume of blood in the fluid is calculated according to the formula b :

[0069] V m = V b x Hct x η

[0070] Embodiment 6. A container comprising at least two inlets, an RBC flocculating agent, and a volume capacity of about 100 ml, about 250 ml, about 500 ml, about 1000 ml, about 1200 ml, or about 2000 ml.

[0071] Embodiment 7. The container according to embodiment 6, wherein the RBC flocculating agent is polyDADMAC in an amount of about 0.3% to about 0.75% and the volume capacity of the container is about 1200 ml.

[0072] Embodiment 8. The container according to embodiment 6, comprising a series of calibration blood volume markings, wherein the markings do not correspond to a liquid volume measurement of the container.

[0073] Embodiment 9. The container according to embodiment 6, comprising a lid having at least one inlet adapted to receive a straw and a second port adapted to apply a vacuum draw in the container.

[0074] Embodiment 10. A collapsible fluid collection container comprising an RBC flocculating agent, a series of calibration blood volume markings, at least two ports, a mounting ring, and a volume capacity of about 250 ml, about 500 ml, about 750 ml, about 1200 ml, or about 2500 ml.

[0075] Embodiment 11. The collapsible fluid collection container according to embodiment 10, wherein the RBC flocculating agent comprises polyDADMAC.

[0076] Embodiment 12. A blood indicating panel for estimating the amount of blood in a fluid, the panel comprising a series of calibration indicia corresponding to approximate blood amounts in a liquid containing mammalian blood.

[0077] Embodiment 13. The blood indicating panel of embodiment 12, wherein the calibration indicia are calculated from a volumetric measurement of RBCs settled within a collection device in the presence of an RBC flocculant, a RBC fill rate determined by the fluid collection device, and a mean hematocrit value for the target animal.

[0078] Embodiment 14. The blood indicating panel of embodiment 13, wherein the RBC flocculant comprises polyDADMAC.

[0079] Embodiment 15. The blood indicating panel of embodiment 13, wherein the series of calibration indicia do not indicate a volumetric measurement of the liquid in the collection device.

[0080] Embodiment 16. The blood indicating panel of embodiment 13, wherein the series of calibration indicia provide approximate values of blood amounts in a fluid material collected in the collection device from an adult human, the blood indicating panel calibrated to be consistent with a mean hematocrit of blood from the adult human and a RBC fill rate determined by the collection device for a collection device having a volume of about 1200 ml.

[0081] Embodiment 17. The blood indicating panel of embodiment 12, wherein the animal is a horse, a human, or a cow.

[0082] Embodiment 18. The collapsible fluid collection container of embodiment 10, comprising a bag having a volume of about 1200 ml and adapted to be placed in a 1200 ml collection jar.

[0083] Embodiment 19. The collapsible fluid collection container of embodiment 18, wherein the bag comprises a blood indicating panel comprising a series of indicia calibrated to provide a visual estimate of the amount of blood in a liquid contained in the bag.

[0084] The following examples are provided to illustrate preferred embodiments of the present application.

[0085] Example 1 - RBC Flocculants

[0086] This example shows materials that can be used as RBC flocculants in fluids containing or suspected of containing blood. The following chemicals in Table I can be provided as RBC flocculants.

[0087] Table 1

[0088]

[0089] Lab testing and downward selection:

[0090] All the identified chemicals listed in Table I were tested and their suitability assessed. Whole bovine blood, purchased from Innovative Research (Novi, MI), was used to determine the suitability of each of the above chemicals as RBC flocculants. The whole blood contained sodium citrate as an anticoagulant. The whole blood was diluted with physiological saline to provide the following blood concentrations tested in this study: 20%, 30%, 40%, 50%, 65%, and 80%.

[0091] Gelatin induces RBC aggregation but does not cause sedimentation. Ultimately, gelatin transforms the entire blood / saline mixture sample into a gel structure. Gelatin is unsuitable for this application without separating RBCs from plasma and saline.

[0092] Dextran 80+CaCl2 also converts blood / saline mixture samples into gels instead of separating RBCs from plasma and saline.

[0093] Acid treatment of blood / saline mixture samples was performed by adding 1.8% of a high-concentration acidic 6N HCl. This brought the sample pH to approximately 4.5. Acid treatment caused RBC aggregation and sedimentation. However, using relatively large amounts of strong acid is not cost-effective for this application, and strong acids are difficult to handle in the process. Therefore, acid treatment can be used to increase ESR.

[0094] PEI is a positively charged polymer that induces the precipitation of RBCs from plasma and saline. PEI must be dissolved under acidic conditions (i.e., pH < 7.0). In other words, at neutral pH, a blood / saline mixture sample must first be acidified to lower the pH before PEI can be used to promote RBC precipitation in blood-containing biofluids. The relative effectiveness of PEI and polydADMAC was compared. PEI-induced ESR (erythrocyte sedimentation rate) was found to be lower than that induced by polydADMAC. PEI is an acceptable flocculant for RBCs in blood-containing biomaterials (liquids).

[0095] PAM is a polymer that can be negatively or positively charged. Negatively charged polymers do not induce the aggregation and sedimentation of negatively charged RBCs. Positively charged PAM can be expected to be used in this application.

[0096] Alum is a positively charged molecule. It does promote lower ESR, but at a very low rate and in very low amounts. The relative molecular weight of the flocculant is considered when choosing a suitable flocculant, as it enhances the settling of RBCs due to gravity. In studies using PEI and polyDADMAC, it was found that those higher molecular weight polymers resulted in faster RBC settling.

[0097] PolyDADMAC is another positively charged polymer. It is also water soluble at neutral pH. PolyDADMAC has the ability to rapidly induce RBC sedimentation in the presence of blood / physiological saline mixture samples. Only small amounts of polyDADMAC are needed to induce sedimentation. Quantitative studies were conducted to determine the proportion of polyDADMAC needed in proportion to the amount of blood in a biological mixture containing blood. These studies also suggested that an excess of polyDADMAC can hinder or even stop RBC sedimentation. This can be because the RBCs are coated and surrounded by the positively charged polyDADMAC, so the coated RBCs repel each other by positive charge rather than negative charge, thus hindering and / or preventing aggregation and sedimentation.

[0098] Low cost, high molecular weight (at least 100 KDa, 200 KDa, 300 KDa, or about 400 KDa), positively charged polyDADMAC was identified as the preferred, cost effective flocculant for rapidly inducing RBC sedimentation. Other high molecular weight, positively charged polymeric flocculants (e.g., PEI, PAM, etc.) can also be used in the practice of the methods of the present invention and in the formation of the containerware. Acid treatment is also contemplated as part of the method of providing RBC sedimentation.

[0099] Example 2 - Blood loss estimation

[0100] This example demonstrates the material polyDADMAC as an effective flocculant for RBCs contained in a fluid, and the use of this flocculant in estimating the total amount of blood in a fluid containing blood and other liquids, including physiological saline.

[0101] High molecular weight (at least 100, 200, 300, or 400 KDa) research grade polyDADMAC and industrial grade polyDADMAC were obtained from Sigma-Aldrich (catalog numbers 409022 or 409030). The industrial grade product is commercially available from Kemira Chemical under the trade name "Superfloc C-591" which contains a 20% concentration of polyDADMAC. This material is used for water treatment. Studies were conducted to compare the effectiveness between the research grade and industrial grade products. The studies showed that Superfloc C-591 produced similar results in RBC sedimentation as the research grade product. The industrial grade product is less expensive than the research grade product. The purity of C-591 is different, resulting in some differences in RBC flocculation. When using both the research grade and industrial grade polyDADMAC, a 20 wt.% stock solution of water was diluted with water to a 6.67 wt.% working solution to reduce the viscosity of the solution and enhance handleability. The dry weight of flocculant used for a 1200 ml collection jar was about 600 mg of polyDADMAC.

[0102] The amount of flocculant required to promote RBC settling. Superfloc C-591 (obtained from the manufacturer (viscous liquid, 20 wt. % in H20)) was used as the RBC flocculant to test the bovine blood and saline mixture. The amount of flocculant required to promote a faster RBC settling rate was tested. It was found that only about 0.4% (v / v %) of the Superfloc C-591 working solution added to the total blood / saline mixture volume was required to provide acceptable RBC fast (within 15 minutes) settling in the mixture. For example, in a 1 liter blood / saline mixture sample, only 4 ml of the Superfloc C-591 working solution was required regardless of the blood concentration in the mixture. This translates to about 320 mg of the polyDADMAC flocculant for a 1.2 liter canister.

[0103] Figure 1 Three 50 ml volume blood / saline mixture samples were shown with blood concentrations (from left to right in the figure) of 20%, 40%, and 65% respectively. The addition of the same amount of 200 μΐ (or 50 ml of 0.4%) Superfloc C-591 working solution induced red blood cell settling at all of these blood concentrations. However, as discussed below, it was found that the settling rate differed depending on the blood concentration in the particular mixture.

[0104] The tests also showed that lower or higher concentrations of the Superfloc C-591 working solution could be used. For example, concentrations as low as 0.3% (v / v %) and as high as 0.6% (v / v %) were found to be effective, however the exact upper and lower limits were not determined. It was found that the addition of a 6% (v / v %) concentration of the Superfloc C-591 working solution to a blood / saline mixture sample containing a 20% blood concentration induced minimal settling, but this can not be the upper limit of settling.

[0105] The aspect ratio of the blood container (receptacle or canister). During the experiments, it was found that the RBC settling rate was partially dependent on the aspect ratio D:H of the container, where D is the diameter and H is the height of the container, for example as shown in Figure 2 The aspect ratio D:H depends on the shape of the container. When D:H is higher, the settling rate is higher because the larger D provides a larger area or space for the aggregated RBCs to settle by gravity. Therefore, the settling rate can be different when different blood collection containers (or canisters) are used.

[0106] Another finding during the experiment was that Superfloc C-591 flocculant caused RBCs to settle when it was added to the container before the blood / saline mixture sample was added, and when it was added to the container after the blood / saline mixture sample was already in it.

[0107] Sedimentation rate and blood concentration. Current research shows that blood concentration in the blood / saline mixture also affects the sedimentation rate of RBCs, with samples having lower blood concentrations showing faster sedimentation rates compared to samples with higher concentrations. For example, Figure 3A , 3B The sedimentation rates of RBCs in 30%, 40%, and 65% blood / saline mixture samples were elucidated by 3C and 3C, respectively.

[0108] Graduated glass cylinders were used in these studies. Figure 3D To determine the differences in sedimentation rates, three groups of 60 ml blood / saline mixture samples were used in the experiment, with blood concentrations of 30%, 40%, and 65%. First, 240 μL (or 60 ml of 0.4%) Superfloc C-591 working solution was added dropwise to a glass graduated cylinder. Then, 60 ml of the blood / saline mixture sample was added to the glass graduated cylinder. The separation of RBCs was visually assessed by the clear distinction of the fluid layers within the glass graduated cylinder, where the sedimenting RBCs were a less transparent fluid layer towards the bottom of the cylinder, while the plasma / saline layer was a more transparent fluid layer above the sedimenting RBCs. The volume of sedimenting RBCs in the graduated cylinder was recorded using the graduated cylinder graduations, and the data was recorded every minute.

[0109] At time 0, the sedimentation volume of RBCs was set at 60 ml because no visible separation was observed at time 0. After time 0, RBCs began to settle from the plasma / saline layer and initially settled towards the bottom of the container at a high sedimentation rate, but the sedimentation rate decreased over time. In this respect, as the amount of RBCs separating from the plasma / saline layer increased, fewer RBCs separated from the plasma and saline. Eventually, the RBC sedimentation rate became very low, so the rate of change in RBC volume was less than 0.5% / min. At this point, the sedimentation volume of RBCs was considered stable. In other words, stable sedimentation was achieved when the change in the volume of sedimenting RBCs was less than 0.5% / min. “RBC hematoma” is denoted as V. m It is the volume of the RBC layer at the bottom of the container when RBC settling is considered stable.

[0110] Figure 3AThe blood / normal saline sample mixture with 30% blood concentration started RBC sedimentation almost immediately after mixing with the flocculating agent. Most of the sedimentation was completed within one minute. Stable RBC sedimentation was reached at about 10 minutes after the sample was mixed with the flocculating agent. The stable RBC sedimentation volume V m was about 9.5 ml. For the blood / normal saline mixture sample with 40% blood concentration, the sedimentation rate was slightly slower Figure 3B . Most of the sedimentation of the RBC occurred within 2 minutes instead of the 1 minute observed for the 30% blood concentration sample. Stable sedimentation was reached at about 15 minutes for the 40% concentration sample, and the V m was about 14 ml. For the 65% blood concentration sample, the sedimentation was slower Figure 3C . It took over 60 minutes before stable sedimentation was observed. The method of determining the volume of blood in the blood / normal saline mixture worked best and consistently in liquids containing 50% or less blood. The characteristic of fast sedimentation was the achievement of stable sedimentation of the RBC in the normal saline / blood mixture within 15 minutes of adding the flocculating agent at room temperature (the mixture was not stirred during the sedimentation process).

[0111] To ensure that the blood concentration in the fluid remains below about 50%, blood (for optimal RBC separation), additional normal saline, or other appropriate blood diluent can be added. It was found that blood / normal saline mixtures with high concentrations of blood can still achieve fast sedimentation. One way the sedimentation can be restarted is that when a slowdown in the sedimentation rate occurs, additional volumes of normal saline can be added to further dilute the blood. As Figure 4 shown in FIG. 3, three sets of 10 ml blood / normal saline mixture samples with 80% blood (bovine blood containing sodium citrate anticoagulant) concentration were prepared in three bottles. All samples were mixed with 30 μL (about 10 ml 0.3%) of Superfloc C-591 working solution (about 2 mg dry weight of Superfloc C-591). No sedimentation was observed before the samples were diluted. Normal saline was added to each bottle to dilute the blood concentration, Figure 4 from left to right, 5 ml, 10 ml, and 20 ml of normal saline were added to dilute the blood concentration of the samples to 53.3%, 40%, and 26.7%, respectively. The containers were gently stirred to mix the added normal saline with the samples. After dilution, the RBC started to sediment.

[0112] The 26.7% blood concentration mixture and the 40% blood concentration mixture reached stable sedimentation at about 10 minutes. The 53.3% blood concentration mixture reached stable sedimentation at about 16 minutes. As Figure 4 shown in FIG. 4, even with different amounts of normal saline present, the final RBC sedimentation volume V mThe same result was obtained because the initial 10 ml of 80% blood concentration mixture contained the same amount of blood, 8 ml. This study shows that 1) more saline can be added to dilute the blood / saline mixture to help increase the RBC sedimentation rate; and 2) the total blood volume can be estimated by RBC sedimentation because the volume of RBCs that collect at the bottom of the container (i.e., the RBC hematocrit) is not affected by the amount of saline in the blood / saline mixture.

[0113] It should be noted that when using other flocculants or other chemical treatments discussed above, the test protocol previously outlined can be used subsequently to further test to determine the optimal flocculant concentration and blood / saline ratio.

[0114] Repeatability. The RBC sedimentation study using a 40% blood (sodium citrate anticoagulant containing bovine blood) concentration mixture was repeated three times Figure 10A ). The standard deviation error bars are shown at the different points on the line in Figure 10A Figure. The repeatability of the experiment is very good, especially at the points on the line where the sedimentation is near steady. The relatively large variations that occur initially are shown by the longer standard deviation error bars at those points on the line, but these variations are likely due to errors in the visual readings caused by the rapid changes in the volume of the sedimented RBC layer at those points. The data variations are very small when the sedimentation is near steady. In other words, the RBC hematocrit V m varies very little when the sedimentation is steady.

[0115] Total blood volume estimation. To demonstrate the feasibility of estimating the total blood volume, an algorithm was developed to estimate the total blood volume in a blood / saline mixture based on the volume of the sedimented RBCs V m . If the actual volume of RBCs is represented as V c ( determined by centrifugation), the total blood volume is represented as V b , and the patient's hematocrit is represented as Hct, then the following relationship is created:

[0116] η = V m / V c , Hct = V c / V b → V b = V m / (Hct x η)

[0117] Since Hct can be obtained from patient data measured prior to surgery and V m can be measured based on the method described above, the total blood volume V m can be estimated when the value of the fill rate (η) between V c and V b is determined from a pre-experiment (methods and examples are given herein to show how to determine the fill rate η).

[0118] The value of the fill rate η can be different for different blood types (e.g., human vs. non-human). The fill rate value can also be container specific, as it can vary depending on the size and shape (aspect ratio) of the container used to collect the blood. In this regard, recall tests have shown that container shape affects ESR (erythrocyte sedimentation rate). Thus, the fill rate η should remain the same in the case of using the same blood type and container shape for blood measurements.

[0119] The fill rate η is determined empirically by using blood / saline mixtures of known hematocrit values and known blood volumes. Preferably, the value of the fill rate η is determined by obtaining an average fill rate value from a number of blood / saline mixture samples having different known blood concentrations. In this regard, by conducting studies with blood from the same animal species (cattle, horses, humans) and containers of defined size and shape, an appropriate fill rate η can be calculated when different known blood / saline test concentration mixtures are detected in the presence of a flocculating agent as described herein. The more the number of different blood / saline mixtures that are detected, the more statistically reliable the average fill rate value that is obtained. As discussed above, in some embodiments, the blood concentration of the collected fluid should be about 50% or less blood. Additionally, an average hematocrit can be determined from a number of hematocrit values obtained from a representative number of subjects (animals / humans). As known to those skilled in the art, hematocrit can be calculated using conventional capillary tube centrifugation methods. When the blood volume is known during a particular collection, the average hematocrit value for a group of animals (e.g., a group of humans or a group of horses, etc.) will provide a value V c .

[0120] As described above, the value of the fill rate η is preferably an average fill rate value calculated from a group of blood / saline mixture samples in a defined collection container. For a number of individual samples, the actual RBC volume V c promoted in the presence of RBC flocculating agent (by ordinary gravity at room temperature) and the settled RBC volume V m are determined, and an average fill rate value is determined. To determine the actual RBC volume V c , the hematocrit of the blood added to the mixture sample is multiplied by the blood volume added to the sample.

[0121] Next, as described above, the RBC packed volume V m for each mixture sample can be determined using the conventional volume scale markings on the container. That is, in the presence of a flocculating agent, V mAs part of this method, the same type and amount (concentration in total fluid mixture volume) of RBC flocculant should be added to the collection vessel. As shown in Table 2, the fill fraction η remains relatively consistent even as the concentration of the flocculant, polyDADMAC, is varied. Thus, a range of flocculant concentrations (0.3%, 0.4%, and 0.75% flocculant in total liquid volume) can be used to induce RBC settling without significantly affecting the fill fraction. Additionally, the data in Table 2 indicate that the fill fraction is relatively insensitive to the amount of blood in the liquid mixture (blood / physiological saline) when the flocculant concentration is held relatively constant.

[0122] Using the actual RBC volume V c and the settled RBC hematocrit V m , the fill fraction value η can be determined for each sample (i.e., each different blood concentration mixture). Then, an average fill fraction value can be calculated. For example, for the bovine blood, the average fill fraction calculated is 1.61 (see Table 2).

[0123] To confirm the empirical determination of the fill fraction value, the value of the fill fraction η was determined for bovine blood purchased from a commercial supplier, which contained sodium citrate as an anticoagulant. A plastic beaker of labeled volume (ml) was used, and the procedure was as shown in Figure 4 The results are shown in Table 2 below. Prior to the experiment, the average hematocrit Hct was calculated for the bovine blood from a large number of bovine blood samples, and determined to be 37.3% on average. This average Hct was used in the table below, and for the current estimate of the amount of blood in the samples. A conventional capillary centrifugation method was used to determine individual Hct values.

[0124] Table 2

[0125]

[0126] The fill fraction values obtained from these experiments show relatively small differences. In these experiments, the blood concentration of all of the blood / physiological saline mixture samples in the table above was less than 50% because mixtures with higher amounts of blood could be diluted by the addition of physiological saline to provide mixtures with less than 50% blood. After determining that the Hct was 37.3% and the average fill fraction η value was equal to 1.61, the following equation (from Equation 1) can be used to estimate the total amount of blood in a blood / physiological saline mixture:

[0127] V b = V m / (37.3% x 1.61) = 1.67 x V m

[0128] The number 1.67 in the above equation is the calculated value of 1 / 37.3% (average Hct) x 1.61 (average η value from Table 2).

[0129] The following table shows a comparison between the actual blood volume known to be present in each sample and the approximate blood volume (in ml) determined in the sample by using the formula described above. The results confirm that the formula and technique provided herein can be used to provide an approximation of the blood volume in a liquid sample containing mammalian blood (e.g., bovine blood) in the presence of an RBC flocculant (e.g., a polymeric flocculant such as polyDADMAC). Moreover, the data show that the approximate blood volume present in the collected mixed blood / saline liquid sample is closely related to the actual blood volume in the liquid.

[0130] Table 3

[0131] Experiment V m (ml)]]> Approximately V b (ml)]]> Actual V b (ml) 30 ml 20% blood 3.5 5.85ml 6ml 30 ml 40% blood 7.5 12.53ml 12ml 50 ml 20% blood 6.0 10.02ml 10ml 50 ml 40% blood 12.0 20.04ml 20ml

[0132] The 20% blood recorded in Table 2 was composed of 200 ml of bovine blood and 800 ml of saline.

[0133] Example 3 - Fluid collection canisters

[0134] This example shows the preparation of a specific fluid collection container with an RBC flocculant.

[0135] Blood-containing fluid collection container. The fluid collection container used in the following examples was a 1200 ml suction canister as shown in Figure 7B The flocculant, polyDADMAC, was applied to the canister (1) as a film of flocculant particles (2) dispersed on the bottom and walls of the canister.

[0136] Flocculant - polyDADMAC. Kemira's "Superfloc C-591" was used as a source of polyDADMAC. The quality or purity of this product was inconsistent. Therefore, a high molecular weight (200-350 KDa) 20% polyDADMAC from Sigma-Aldrich (Sigma Catalog # 409022) was used. The results indicated that the Sigma version of polyDADMAC significantly improved the test results. However, it is expected that virtually any number of different sources of flocculant could be used in the practice of the present invention as well as in the manufacture of fluid collection containers containing the flocculants described herein and treated with them.

[0137] Example 4 - polyDADMAC as a flocculant for a 1200 ml collection canister for optimization of blood volume approximation for a mixture containing blood

[0138] In the operating room, biological fluid waste collection canisters are used to collect a volume of fluid that will include an unknown volume of blood. The amount of blood in a 1200 ml canister volume can vary from 10 ml to 1200 ml. The amount of blood in the collected collection fluid can also vary depending on the specific species of animal, the gender and weight of the patient, and the specific medical procedure being performed. However, the amount of blood in the fluid collected in the collection canister during a typical adult surgical procedure usually contains from about 20% to about 50% blood. The conventional techniques used in a standard hospital operating setting for blood volume determination only provide a gross estimate of the blood volume that is at least 50%-75% inaccurate most of the time and is not available until several hours after the surgical procedure is completed.

[0139] In most cases, the amount of blood in the fluid aspirated during a surgical procedure is 50% or less. In those cases where the fluid contains more than 50% blood, the method and apparatus of the present invention can be used to accurately determine the blood volume by adding saline or other diluent to the fluid to reduce the blood concentration to facilitate the settling of the RBCs in the fluid in the presence of the RBC flocculating agent, polyDADMAC.

[0140] This example demonstrates that a relatively constant amount of RBC flocculating agent, such as polyDADMAC, can be used to achieve a relatively accurate blood volume estimate for fluids containing about 50% or less blood concentration. This is achieved by using a visual reading of the settled volume of RBCs in a calibration canister containing the RBC flocculating agent. This RBC settled volume is then used in a calculation to determine the blood volume in the mixture. The settled RBC volume value alone is not sufficient to accurately estimate the blood volume in the mixture.

[0141] Three different volumes of blood / saline mixtures containing 40% blood were tested. The volumes of the 40% blood / saline mixtures tested were: 200 ml, 800 ml, and 1200 ml. A Sigma-Aldrich polyDADMAC (containing 20 wt.% water) solution was diluted with saline to provide a working solution containing 6.67 wt.% polyDADMAC in water. This working solution was used to provide the approximate amount of flocculating agent in this study.

[0142] Table 4 provides the optimal flocculating agent dose for each volume of 40% blood / saline mixture tested. The optimal flocculating agent dose was determined as the amount of flocculating agent required to provide the fastest rate of settling of red blood cells out of the mixture. The RBCs in each of the mixture volumes tested reached a visually discernible level of settling in the canister, where a relatively clear fluid was observed above the visually discernible settled RBC meniscus about 15 minutes after the blood / saline mixture was combined with the flocculating agent at room temperature. The rate of settling was observed to vary depending on the amount of flocculating agent provided in the mixture.

[0143] According to 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 of polyADMAC) is optimal for facilitating the rapid settling of RBCs in a 100 ml volume of blood / physiological saline mixture. For a larger 1200 ml jar having a volume of about 1200 ml, about 9 ml of polyDADMAC flocculant working solution (or about 600 mg dry weight of polyDADMAC) will be provided on the bottom of the jar or on the jar walls to facilitate the rapid settling of RBCs in a blood / physiological saline mixture having a volume of up to 1200 ml. In the following studies, about 0.75 ml to about 1.5 ml of flocculant working solution is used per 100 ml (or 0.75% v / v) of blood / physiological saline mixture. This concentration value is slightly greater than the 0.4% to 0.6% used in the studies described herein when using the industrial grade flocculant Superflock C-591 version of polyDADMAC.

[0144] Based on the identified 0.75% v / v polyDADMAC concentration, a 1200 ml jar collecting up to 1200 ml of blood-containing fluid will require about 9 ml of polyDADMAC working solution (or about 600 mg dry weight of polyDADMAC).

[0145] Table 4

[0146]

[0147] Table 4 shows the amount of flocculant that achieves optimal RBC settling for different volumes of 40% blood / physiological saline mixture. As shown, 9 ml of RBC flocculant polyDADMAC (about 600 mg dry weight) provides optimal RBC settling in a 1200 ml volume of blood physiological saline mixture.

[0148] Example 5 - Method of preparing a flocculant-treated jar

[0149] This example describes various methods in which a flocculant can be provided and distributed within and / or on a fluid collection container, particularly a blood-containing fluid. Although a specific polymeric flocculant, polyDADMAC, is used in this example, many other polymeric and non-polymeric flocculants can be used in the practice of the present invention to provide the methods and devices described herein.

[0150] Vertical band coating on jar walls. A 1200 jar was coated with a 9 ml volume of material containing 600 mg of flocculant. The jar was coated in the form of a vertical band 1” wide in the jar. In this way, only the flocculant that is dissolved or released into the blood / physiological saline mixture when immersed in the mixture can cause RBC settling. In other words, the vertical band of polyDADMAC coating can provide a controlled release of flocculant proportional to the volume of blood / physiological saline mixture.

[0151] Two methods were used to coat the vertical band with the coating. One was to use a brush to provide the 1" band directly on the can wall. The other method was to coat the flocculant solution on a 1" wide clear adhesive tape and then stick the clear tape on the can wall. The can had a hydrophobic surface making it difficult for the flocculant solution to stay on the wall. An ozone treatment was developed to change the hydrophobic can wall to hydrophilic. This technique was used to improve the flocculant adhesion.

[0152] After the vertical flocculant band was coated, the can was tested with commercially available bovine blood from a commercial supplier. The can with the flocculant band did promote RBC settling. However, the settling rate was slightly slower. It took over 20 minutes for the RBC to settle at the bottom of the can. Typically, a faster RBC settling rate is desired. While not intending to be bound by any theory or specific mechanism of action, the slower rate can be related at least in part to the time required for the flocculant coating to dissolve and diffuse throughout the blood / physiological saline volume. It takes more time for the flocculant to diffuse from the single band coating to the entire blood / physiological saline mixture. A thinner coating distributed throughout the can is expected to speed up the dissolution and distribution of the flocculant.

[0153] A ultrasonic atomization technique was used to coat uniformly over the entire can. An ultrasonic based method was used to coat the inside of the blood collection can. The ultrasonic atomization coating is a low pressure, low velocity (typically on the order of 3-5 inches per second) coating that is distinguished from a spray coating. A piezoelectric transducer converts electrical input into mechanical energy in the form of vibrations. High frequency acoustic vibrations atomize the liquid into a fine mist spray Figure 6 ). The low pressure, low velocity spray significantly reduces overspray as the droplets tend to settle on the substrate rather than bounce off the bottom. The spray pattern can be precisely controlled and shaped. Spray patterns as small as 0.070 inches wide to as large as 1-2 feet wide can be produced using these types of specialized spray shaping equipment. The atomization device used in this method has a 60 kHz ultrasonic nozzle.

[0154] Figure 7A A manual coating process is shown for a can using an ultrasonic atomizer. The atomizer tip is moved along the can wall, starting at the bottom of the can and moving gradually up to the top of the can. The misty spray coated area changes the clear wall to a misty surface, which guides the manual coating process to cover the entire can wall and make the coating as uniform as possible based on visual observation. An automated coating method can be developed in the future to make uniform coating.

[0155] Different concentrations of polyDADMAC were tested and high vaporization alcohols such as methanol and ethanol were incorporated into the spray solution to speed up the drying process. Using an ultrasonic atomization device with a strength setting of "10", the polyDADMAC working solution (prepared in DI (deionized) water) provided the best misty spray to settle the flocculant Figure 7A). The syringe pump was set to 60 ml / h. Approximately 4.5 ml of flocculant coating solution was needed to coat the entire 1200 ml of tank wall. Therefore, to coat 9 ml of flocculant solution onto the tank, two batches of flocculant coating were required. The 9 ml volume of flocculant solution contained approximately 600 mg of polyDADMAC.

[0156] Between each coating, the tank was allowed to dry completely in the oven for several hours or at room temperature for 24 hours. Figure 7B A tank is shown with two batches of coating applied to its entire wall. The use of a more powerful ultrasonic atomization device would allow for a higher concentration of the coating solution. In this way, a single batch of coating could be used to deliver a 4.5 ml solution containing all of the polyDADMAC instead of a 9 ml volume to provide the flocculant concentration described above.

[0157] The flow scheme provides the manual steps to prepare the flocculant treated (coated) tank:

[0158] Preparation of the coating

[0159] 1. Prepare the polyDADMAC working solution by mixing one part of 20 wt. % polyDADMAC (Sigma Catalog # 409022) with two parts of DI water.

[0160] 2. Fill a 60 ml syringe with the polyDADMAC working solution.

[0161] 3. Set the intensity of the ultrasonic atomization device to "10".

[0162] 4. Set the syringe pump rate to 60 ml / h and the delivery volume to 4.5 ml.

[0163] Manual coating method

[0164] 1. Turn on the ultrasonic atomization device and begin moving the ultrasonic nozzle from the bottom of the tank to coat a misted spray on the tank wall and bottom.

[0165] 2. Gradually move the ultrasonic nozzle from the bottom to the top of the tank along a spiral path and ensure that the wall is coated with polyDADMAC everywhere. The area coated with the polyDADMAC mist shows a misty appearance as shown in the figure below.

[0166] 3. Typically, 4.5 ml of the diluted polyDADMAC is sufficient to provide one layer of coating to the entire tank.

[0167] 4. Allow the coated tank to dry overnight at room temperature.

[0168] 5. After the first layer of coating has dried, apply a second layer to the tank in the same manner as described above. After two coatings, a total of 9 ml of diluted polyDADMAC has been applied to the tank.

[0169] 6. The canister is dried again overnight and then ready for use.

[0170] Example 6 - Estimation of the fill rate (η) of a polyDADMAC coated 1200 ml canister

[0171] A polyDADMAC coated 1200 ml canister was prepared using a canister with a volume of 1200 ml and having the dimensions of a bio-waste canister used in an operating room. A typical configuration of such a canister in an operating room environment is provided in Figure 5. First, the fill rate η associated with this coated canister was determined.

[0172] In this study, bovine blood was used that was commercially available from a commercial supplier. The blood was refrigerated and then warmed to 23°C at the time of testing. In addition, the bovine blood contained sodium citrate to prevent clotting.

[0173] The bovine blood and physiological saline were added to the flocculant treated canister to obtain a determined blood / physiological saline ratio. To simulate the process of providing a blood containing liquid into a collection canister during routine operations, for example, a 600 ml volume of blood / physiological saline mixture (40% blood) was transferred into the flocculant treated canister according to the following technique. Container A was prepared to contain 240 ml of blood and container B was prepared to contain 360 ml of physiological saline. A serum pipette was used as the aspiration probe. The aspiration probe was connected through tubing to the inlet (patient port) of the canister. The second port of the canister was used to further connect the canister to a vacuum line and was used to create a vacuum in the canister. Under vacuum, the pipette was placed into the container containing the blood or the container containing the physiological saline to selectively aspirate the respective fluid into the canister containing the flocculant (polyDADMAC) Figure 8 ). Thus, a total fluid volume of 600 ml of 40% blood was provided in the canister.

[0174] After all the blood and physiological saline was aspirated into the third canister, the mixture was monitored to assess the separation / settling of the RBC from the plasma and physiological saline in the presence of the flocculant. The volume of the RBC settling line was recorded every minute for 20 minutes.

[0175] Studies were performed using different volumes of 40% blood / physiological saline mixture solutions (200 ml, 400 ml, 800 ml and 1200 ml). Each study was repeated three times. Figures 10A-10D The change in RBC settling volume over a 20 minute time period using a 40% blood mixture is shown. The study was also performed three times with 20% blood / physiological saline mixture solutions in 200, 400, 800 and 1200 ml volumes Figures 9A-9D ). To obtain an average fill rate η applicable to different volumes of fluid collected in this canister and different blood concentration mixtures, both 20% and 40% blood concentrations were tested as well as four different volumes from 200 ml to 1200 ml.

[0176] Using a 20% blood mix (20% blood / 80% saline), RBCs settled very rapidly in flocculant treated jars. A 30 ml (V m ) RBC sedimentation Figure 11A ) was visually observable within 10 to 15 minutes at room temperature. The average V m and calculated V ic for three replicate experiments are shown in Table 5.

[0177] These experiments demonstrate that the polyDADMAC coating facilitates rapid RBC sedimentation out of blood / saline mixtures within 15 minutes at room temperature. Without flocculant, RBC sedimentation in these blood / saline mixtures would take 3-6 hours at room temperature.

[0178] Estimate the fill rate η. Using data from the above experiments, the fill rate η (see Equation 1) between the volume of settled RBCs (V m ) and the actual RBC volume (V c ) can be determined empirically. Table 5 shows the empirically determined fill rates for 1200 ml Medi-Vac jars, with an average of 1.20. Since the fill rate encompasses a large volume range of 200 to 1200 ml and a large blood concentration range, the variability of the fill rate is relatively high, above 13%. This variability in fill rate will also affect the variance of the blood loss estimate. The following fill rate η values are calculated for each respective blood mix. Then the average η is calculated.

[0179] Table 5 Empirically determined fill rates

[0180]

[0181]

[0182] In this study, the average hematocrit value for the bovine blood used was 35.4% and the average fill rate η derived was 1.2. Using the formula in Equation 1, the following formula was created to estimate the blood volume in the fluid collected in a jar containing flocculant:

[0183] V b = V m / 35.4% x 1.2 = 2.35 x V m

[0184] The estimated blood loss values for each blood mix 1-8 are given in Table 6 below and compared to the known blood volume in the sample.

[0185] Table 6

[0186] Blood mixture V m (ml)]]> Estimated blood volume (ml) Actual blood volume (ml) 1. 200 ml 20% blood 18 42.30 40 2. 400 ml 20% blood 30 70.50 80 3. 800 ml 20% blood 60 141.0 160 4. 1200 ml 20% blood 100 235.0 240 5. 200 ml 40% blood 30 70.5 80 6. 400 ml 40% blood 70 164.5 160 7. 800 ml 40% blood 160 376.03 320 8. 1200 ml 40% blood 233.3 548.3 480

[0187] As shown in the above table, the amount of blood loss calculated using the present formula correlates with the actual amount of blood in the fluid. Thus, the method and apparatus of the present application prove to be a tool for providing a physician / healthcare professional in a surgical environment with a simultaneous visual indication of the amount of blood loss during a surgical procedure that is more accurate than traditional methods (saline bag usage assessment and / or post-operative assessment based on total patient fluid acquisition).

[0188] Based on the observed RBC sedimentation volume (in ml) in a graduated jar in the presence of a flocculating agent, without any electrical, temperature or other material manipulation procedures, a blood indicator panel is designed using the above formula that provides a direct visual tool for the approximate total amount of blood in the collected bodily fluid.

[0189] Example 7 - Creation of a Blood Volume Indicator Panel for Blood Volume Assessment in a Biological Fluid Container

[0190] In order to allow a user to easily estimate the amount of blood by visually detecting the sedimented RBC in a container (e.g., a container containing a flocculating agent), a blood volume indicator panel is provided with a graduated scale for a fluid collection container, and indicates the approximate total amount of blood in the collected fluid based on the level of RBC sedimentation in the presence of a flocculating agent in the collection container. It is contemplated that these collection containers can or can not include traditional volumetric measurements.

[0191] In order to create the graduated scale for the blood volume indicator panel for the present container (jar, etc.), the following Equation 2 is used:

[0192] V m = V b x Hct xη2)

[0193] Equation 2 will employ an average Hct calculated from multiple animals / humans of the same species and approximately the same age, and the same gender. For example, for an adult male, the average Hct is approximately 45%.

[0194] In this study, the following formula is used to create a blood indicator panel for large mammals:

[0195] Equation 3:

[0196] V m = 0.43 x V b

[0197] This formula will be used, and the average Hct for bovine blood is 35.4%, and the average fill factor η = 1.20, as calculated for bovine blood (Table 5).

[0198] A 50 ml estimated blood volume marker is provided on the blood volume indicator panel that corresponds to a visually discernible RBC sediment volume of about 21.5 ml. Equation 3 (see Table 7) is used. A 100 ml estimated blood volume calibration marker can be generated on the blood volume indicator panel that corresponds to a 43.02 ml RBC sediment volume line of the container, and so on.

[0199] The scale markings of the blood volume indicator panel provide a series of visually identifiable markers that are not related to the volume measurement of the material in the jar, but are related to the approximate blood volume in the fluid collected in the jar. Figure 15 An example of a typical blood indicator panel (BIP) is provided (see left Figure 6 calibration markers at 50 ml, 100 ml, 200 ml, 400 ml, 600 ml). Including a blood indicator panel on a conventional collection container with standard volume markings (see Figure 16, right Figure 1 ), an immediate visual estimate of the blood volume in the collected fluid can be provided without the need for mathematical calculations or other manipulation of the collected or settled material. As shown, the blood volume identified with the calibration markers of the BPI does not coincide with the conventional RBC sediment volume in the fluid. Rather, the RBC sediment volume is used as part of the calculation, along with the hematocrit and a defined aspect ratio, to provide an approximate blood volume. Without a flocculating agent, the blood volume in the liquid cannot be estimated in about 3-6 hours because, among other things, the RBCs do not begin to settle until 3-6 hours. Moreover, the presence of a flocculating agent alone, although it promotes rapid RBC settling, does not allow for an immediate estimate of the blood volume in the liquid. As demonstrated in the present results, the sediment volume of the RBCs in the liquid is less than about 50% of the known actual blood volume contained in the test fluid containing a known amount of blood. In the presence of a flocculating agent, the RBC sediment volume must be further corrected for the average hematocrit and packing fraction of blood to provide an approximate blood volume in the liquid.

[0200] BIP panels are created based on derivation of mean hematocrit (Hct), e.g., mean Hct for humans, bovines, equines, etc. To correct for individual patient / animal Hct differences, e.g., individual Hct differences due to gender, species, age, etc., the approximate blood volume value indicated on the BIP can be adjusted by a factor that corrects for significantly higher or lower individual hematocrit values. For example, if a patient has a measured Hct value that is lower, e.g., 80% of the typical Hct for an adult male, then the blood volume indication on the panel observed for that patient will be divided by 80% to provide a closer approximation of the estimated blood volume in the container. More specifically, if the blood volume indication on the panel is 50 ml according to the scale markings on the panel, then the actual blood volume in the biological material collected from the patient will be calculated to be 62.5 ml, where the patient's Hct is 80% of the typical Hct value. Similarly, if a patient has a measured Hct value that is higher, e.g., 110% of the typical hematocrit value for an adult male, then the blood volume indication on the blood volume indication panel will be divided by 110%, which will result in a lower blood volume. For example, if the blood volume indication on the blood volume indication panel is 100 ml, then the actual blood loss volume for the patient will be calculated to be 90.9 ml to correct for the patient's Hct being higher than average (e.g., 10% higher). Figure 8 A 1200 ml canister with a blood volume indication panel is shown.

[0201] A specific blood indication panel placed on a collection canister is shown in Figure 12 The BIP is created to provide an estimate of blood volume in a volume of 20% blood / saline mixture or 40% blood / saline mixture (using bovine blood). The blood indication panel can also be used to assess human blood volume in a fluid sample. This is because both bovines and humans are mammals and blood from bovines and humans share many characteristics, including similar mean hematocrit.

[0202] Table 7. Example blood indication panel markings for a 1200 ml canister. These calibrated blood volume markings correspond to an estimate of blood volume contained in a fluid sample, which is compared in the table to the corresponding RBC sediment volume (indicated by the value V m Bovine blood with sodium citrate

[0203]

[0204]

[0205] The study used RBC flocculant, polyDADMAC coated biofluid collection canisters (1200-ml) to successfully develop a BIP prototype. This size of canister was used in human patients: adult and pediatric operating rooms. Evaluation of the prototype using bovine blood showed rapid settling of RBCs in the presence of the exemplary RBC flocculant and a stable RBC sediment was achieved in 20 minutes. A specially designed calibration mark on the BIP was designed for this collection canister and it can be used to provide a visual estimate of the total blood volume in the collected mammalian fluid. If the patient's measured hematocrit is different from the typical hematocrit used to create the calibration BIP mark, the calibrated blood volume can be corrected to accommodate the individual's blood hematocrit percentage.

[0206] The following provides the average hematocrit for adult males and adult females:

[0207] Normal Hct values: Males - 42-52% (average Hct, 47); Females - 37-47% (average Hct, 42).

[0208] Example 8 - Preparation of 100 ml canister with flocculant

[0209] A 100 ml canister was prepared to contain approximately 50 mg of flocculant. In this example, the RBC flocculant used was polyDADMAC. The flocculant was provided as a volume of the polyDADMAC working solution described herein.

[0210] In a typical operating room environment, smaller volumes of fluid containing blood and other materials (tissue, urine, non-blood, etc.) can be aspirated from the surgical field. The aspiration of these fluids results in blood loss that is not easily measured by the patient. Smaller containers can be prepared in accordance with the present application to accommodate the blood loss estimation in these small, sometimes marginal volume, collection fluids. Thus, these 100 ml containers containing RBC flocculant (e.g., polyDADMAC) are provided and are particularly useful in determining the blood volume in small amounts of collected fluid. These devices can be used, for example, in pediatric applications (infants) as well as low volume marginal fluid collection procedures.

[0211] The 100 ml collection device has an aspect ratio of 0.96. The small 100 ml container containing RBC flocculant was used in the study described in Example 9.

[0212] Example 9 - Fresh blood loss estimation

[0213] This example is provided to demonstrate the utility of the method and apparatus for estimating blood loss in a fluid containing fresh mammalian blood (without anticoagulant). In this example, the non-blood material present in the fluid is physiological saline. This example tests the technique for using a volume of fresh, never refrigerated, mammalian blood for blood estimation. Furthermore, the blood does not contain calcium citrate or any other anticoagulant. In this study, fresh blood samples were obtained from adult horses. Thus, the apparatus and method are particularly suitable for estimating blood loss in mammals, including humans and veterinary animals (horses, dogs, cats, cows, bulls, sheep, pigs, etc.).

[0214] In this example, blood was collected from a live adult horse (approximately 12 years old, weighing 1,200 pounds) that had no known clinical pathologies and was not undergoing any known drug therapy. This animal was undergoing lameness therapy and was receiving a nerve block to control pain. Unlike blood collected from commercial suppliers that contains an anticoagulant such as sodium citrate (used in previous examples), there was no anticoagulant or other drug present in the blood collected from the horse used in this study.

[0215] A total of twelve (12) jars having a total volume capacity of 100 ml were used in this study. The jars were marked with demarcations at 50 ml and 100 ml increments along the side of the jar. The calculated aspect ratio D:H (diameter vs. height) of the 100 ml container was approximately 0.96. In comparison, the aspect ratio of a 1200 ml jar was approximately 0.61. Generally, the greater the aspect ratio of a collection device, the faster any blood contained within the liquid collected will settle out of the collection device. Thus, under similar conditions, the rate of RBC settling in a 100 ml collection device is expected to be faster than the rate of RBC settling in a 1200 ml jar in the presence of an RBC flocculant.

[0216] The 100 ml dry jars were charged with 50 mg of RBC flocculant polyDADMAC, research grade (Sigma catalog # 409022). (See Example 7). Other RBC flocculants, as well as industrial grade versions of these flocculants (including PEI / PAM, etc.), can be expected to be useful in this method and apparatus.

[0217] The indicated amounts of fresh horse blood and physiological saline in Table 8 were then added to each jar, and the RBC settled volume was recorded every minute for 20 minutes:

[0218] Table 8 - Fresh Horse Blood Study: Rate of RBC Settling

[0219]

[0220]

[0221] ND = not detectable by visual inspection

[0222] Blood was drawn from adult horses and a volume of fresh blood (body temperature) was added to each jar in the amounts described above. The top of each jar was then placed in position and the contents were mixed to ensure proper mixing of the saline, blood, and flocculating agent. Each jar was allowed to sit at room temperature and observed. RBC sedimentation times were observed and recorded at 1 minute intervals for 30 minutes. Figure 18 RBC sedimentation rates were observed for various mixtures. No visually detectable RBC sedimentation was observed with the blood mixtures without flocculating agent. In contrast, RBC sedimentation was observed rapidly in all blood mixtures containing flocculating agent within 5 minutes at room temperature.

[0223] Table 9 shows hematocrit values for various large animals. Values for mean corpuscular hemoglobil (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), and packed cell volume (PCV) can be used to provide appropriate custom blood indicator panels and blood volume estimation methods as described in the present disclosure.

[0224] Table 9 - Normal values for red blood cell data for ruminants and horses

[0225]

[0226]

[0227] (MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PCV, packed cell volume)

[0228] Table 10 - Normal values for white blood cell data (adult animals)

[0229] Biological component Cow Sheep Goat Horse White blood cells (xlO 3 / μl) 4-12 4-12 4-13 5.4-14.3 Neutrophils (xlO 3 / μl) 0.6-4 0.7-6 1.2-7.2 2.3-8.6 Bands (x10 3 / μl) 0-0.12 Rare Rare 0-1 Lymphocytes (x10 3 / μl) 2.5-7.5 2-9 2-9 1.5-7.7 Mononuclear cells (x10 3 / μl) 0.025-0.84 0-0.75 0-0.55 0-1 Eosinophils (x10 3 / μl)]]> 0-2.4 0-1 0.05-0.65 0-1 Alkaline granulocytes (x10 3 / μl)]]> 0-0.2 0-0.3 0-0.12 0-0.29 Neutrophil / Lymphocyte (N:L) 0.3-0.6 0.3-0.7 0.6-3.6 0.8-2.8

[0230] Table 11 - Normal values for hemostasis data for ruminants and horses

[0231]

[0232] (Adapted from Duncan JR et al: Veterinary laboratory medicine, 2nd edition, Ames, Iowa, 1986, Iowa State University Press; and Kaneko JJ: Clinical biochemistry of domestic animals, 3rd edition, New York, 1980)

[0233] Example 10 - Blood volume estimation in fluid using a canister containing fresh horse blood (without anticoagulant) in the presence of a flocculant

[0234] This example was performed with fresh blood drawn from an adult horse (12 years old, approximately 1,200 pounds). The horse blood did not contain any anticoagulant. The horse blood had a hematocrit of 32% to 53%. The horse blood was used immediately after being drawn and it was at body temperature (approximately 101 °F, 38.3 °C) when mixed with normal saline in the presence of the flocculant polyDADMAC (600 mg).

[0235] A 1,200 ml canister was coated using approximately 9 ml of a working solution of polyDADMAC sprayed as a flocculant. Thus, the 1,200 ml canister was uniformly coated with a total of approximately 600 mg of dry weight of polyDADMAC as a flocculant. Since the flocculant was uniformly distributed along the canister wall, the amount of flocculant was released in proportion to the volume of fluid provided in the treated canister.

[0236] The following RBC sedimentation volumes were recorded over time.

[0237] A volume of 400 ml of fresh horse blood (not refrigerated, body temperature, no anticoagulant) was placed in the treated canister. A known volume of 250 ml of normal saline was added to the canister. The total volume of fluid in the canister was 650 ml, providing a 61.5% blood solution. The RBC sedimentation volume (gravity only, no centrifugation) Vm was recorded immediately after mixing until the 30 minute time period.

[0238] After 30 minutes of standing, the canister was manually stirred and then again allowed to stand at room temperature. The canister containing the stirred blood was again observed for evidence of RBC sedimentation at room temperature.

[0239] Table 12

[0240]

[0241]

[0242] After the 30 minute observation period, the canister was manually shaken and then allowed to stand. Resedimentation of the RBCs occurred and observations were made every minute for 30 minutes to obtain the level of RBC sedimentation observed as shown in Table 14.

[0243] Table 14

[0244]

[0245]

[0246] It was demonstrated in this study that the settled volume of RBCs in solutions containing fresh whole blood remained relatively stable at room temperature for up to about 30-40 minutes. By agitation, it appeared that the RBCs in the fresh blood sample resettle to provide a discernible RBC settled volume line V m , the settling was very rapid (3 to 4 minutes versus 16-19 minutes, RBC settled volume of about 250 ml to 300 ml). The actual known volume of fresh blood present in the fluid was 400 ml.

[0247] The experimentally determined average hematocrit for the horse blood animal type (42.5%, horse average Hct), Vm (observable RBC settled volume in ml), and the new fill rate value (n) can be used to calculate the total approximate blood volume using the formula m ) and Hct information from multiple horse blood samples can be used to develop a blood volume indicator panel for large animals, such as horses, that can be created and provided along the vertical axis of the collection canister to provide a visual blood indicator panel for use with large animals, such as horses. This would provide an approximate blood volume that can be visually discerned in a biological fluid containing horse blood. The development of a blood volume indicator panel for use with processed cans or other containers can be used to provide a visual indication of horse blood loss, particularly to assess horse blood loss more accurately than is currently available. The information and results provided herein can be used by one of ordinary skill in the veterinary arts to develop a horse blood volume indicator panel without more than ordinary and routine experimental optimization trials and errors.

[0248] Example 11 - Human pediatric applications for estimating blood loss

[0249] This example is provided to provide a canister and method for effectively measuring blood loss in pediatric patients. As used in this example, a pediatric patient is defined as an individual under the age of 12 weighing up to 70 to 80 pounds.

[0250] The total blood volume (TBV) in humans is related to body weight. The TBV in children is approximately 75-80 ml / kg, and is higher in the neonatal period (rising from 85 ml / kg at the end of the first month to a peak of 105 ml / kg, then gradually declining over the next several months). Thus, the total blood volume of a 3.5 kg, 2 week old infant would be approximately 350 ml, while the total blood volume of a 10 kg, 15 month old infant would be approximately 800 ml.

[0251] Because the total blood volume of pediatric patients is greatly reduced, it is especially important to provide blood collection and blood loss estimation systems and devices designed to accurately estimate blood loss from the smaller blood volume collected from pediatric patients. Thus, in the case of an infant or neonate, the pediatric blood loss estimation device of the present invention is specifically designed to be made with a container containing a flocculating agent as described herein and a demarcated canister having a total volume capacity of less than 1000 ml, such as about 500 ml or even about 250 ml.

[0252] Massive acute blood loss in pediatric patients can compromise blood circulation, and thus blood loss should be carefully monitored so that the amount of blood loss specific to about 12% TBV of a particular pediatric patient (about 10 ml / kg) can be detected, assuming the child is stable and has a normal hemoglobin (Hb) level at the start of the procedure.

[0253] By way of example, in some embodiments, a suitable pediatric blood loss collection device will have a capacity of 250 ml. For typical pediatric blood loss amounts, the canister will preferably provide an appropriate D:H aspect ratio. The D (diameter) of the device is typically 2 to 3 inches, and the H (height) is about 2 inches to about 3 inches. With these smaller dimensions, the amount of blood loss collected will provide a reasonable rapid but monitorable sedimentation rate of the RBCs so as to alert the attending physician as to whether the amount of blood loss has reached the prescribed transfusion amount to the pediatric patient. Preferably, a sedimentation rate is achieved that provides for the RBCs of the blood collected in the canister to settle within 15 minutes.

[0254] In some embodiments, the 250 ml container has a conical shape Figure 14 ). The flocculating agent will be provided to the container at the time of the surgical intervention event, or can be provided as a pre-treatment to the canister (such as by spray coating).

[0255] The amount of flocculating agent added to the 250 ml collection device is 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.

[0256] For ease of description, the following average total blood volumes for pediatric groups of patients can be used to calculate when 12% or more blood loss has occurred. The average hematocrit values for groups / classes of pediatric patients (premature neonates, full-term neonates, infants) can also be calculated, and average hematocrit value markers provided along one axis of the canister for these groups of patients so as to provide an immediate visual reference for the attending physician or anesthesiologist to reference and compare the hematocrit obtained for the patient undergoing the procedure:

[0257] • Premature neonates 95 ml / kg

[0258] • Full-term neonates 85 ml / kg

[0259] • Infant 80 ml / kg

[0260] The total approximate blood volume can be calculated by the formula using the mean hematocrit Vm (observable RBC sedimentation volume in ml) for a human child or adult male or adult female of a specific body weight range and / or age, and a determined human blood fill rate value (n). Through multiple human blood sample evaluations of RBC sedimentation volume (Vm) and Hct information, a visual blood volume indicator panel can be prepared for human, particularly pediatric human body models, positioned along the vertical axis of the collection canister. This will provide an approximate blood volume that can be immediately visually discerned in a biological fluid containing human blood that contains a volume of less than about 250 ml. One of ordinary skill in the art, given the teachings provided herein, can develop a vertical canister or other container having a blood indicator panel for human blood volume evaluation in a liquid, particularly for evaluating small human blood loss volumes, without more than routine and ordinary amounts of experimentation and error.

[0261] Example 12 - Collapsible processing container for blood loss collection

[0262] This example presents a collapsible plastic-like container (bag) that can be used for biological fluid loss collection and for estimating blood loss. Such a collection device is envisioned to be particularly useful in a combat environment or any other situation where space for medical equipment is limited.

[0263] It is envisioned that the plastic bag container will include an amount of RBC flocculating agent that is suitable for providing the RBC sedimentation and blood loss estimation features described herein. In some aspects, the bag can be placed within a supporting container, such as a box, canister or other structure. The bag can also include a plurality of markings along the vertical axis of the bag corresponding to volume measurements (e.g., milliliters).

[0264] In some aspects, a clear plastic bag having a volume capacity of about 1,000 ml containing about 300 milligrams to about 4,700 milligrams of flocculating agent, such as polyDADMAC, is placed in the bag. In some embodiments, the bag can include calibration demarcations at 50 ml, 100 ml, 200 ml, 250 ml, 400 ml, 500 ml, 600 ml, 750 ml and 1 liter markers. The bag can also include a BIP, such as in the form of an adhesive strip, that can be placed on the bag and used to provide a visually discernable indication of approximate blood volume in a liquid based on the level of RBC sedimentation in the collection bag / container. Figure 13 An example of this embodiment is provided. Also provided is an insert bag containing RBC flocculating agent and a calibrated human blood BIP designed for use in a 1200 ml collection canister, such as the canister shown in Figure 11A In such an embodiment, the canister itself need not be treated with RBC flocculating agent, rather the insert bag contains the RBC flocculating agent. The insert bag can also optionally include a calibrated BIP for human blood.

[0265] Example 13 - Blood collection kit

[0266] The flocculant containing canisters (1.2 ml, 500 ml, 250 ml, 10 ml) containing blood volume indicator panels can be provided as a kit with a suction tube of a certain length and a tube of a second length suitable for adding saline to the canister and / or a collapsible envelope.

[0267] Instructions can be provided as part of the kit for the end user.

[0268] Example 14 - High temperature aging stability test

[0269] This example demonstrates the stability of the RBC flocculant polyDADMAC in blood containing fluids and the retained activity to provide RBC coalescence (flocculation) after exposure of the polyDADMAC coated canister to high temperatures.

[0270] In this study, the flocculant used was polyDADMAC which was provided as a coating on a canister for the collection of biological fluid materials containing blood components such as collected during a surgical procedure. The coated canisters were incubated at 55 °C for 6 weeks (equivalent to one year shelf life at room temperature). The coated canisters were then compared in a function test after high temperature aging to coated canisters which did not undergo the high temperature aging test.

[0271] Materials:

[0272] 1) Control - Four canisters (Cardinal Health) coated with 600 mg of polyDADMAC were allowed to dry overnight at room temperature (~ 22 °C). Figure 1 2) Experimental - Four canisters (Cardinal Health) coated with 600 mg of polyDADMAC were allowed to dry overnight at room temperature (~ 22 °C). The canisters were then incubated in a convection oven set to 55 °C for six weeks.

[0273] 2) Experimental - Four canisters (Cardinal Health) coated with 600 mg of polyDADMAC were allowed to dry overnight at room temperature (~ 22 °C). The canisters were then incubated in a convection oven set to 55 °C for six weeks.

[0274] 3) Bovine whole blood (Innovative Research Lot #24301) which was stored in the refrigerator and warmed to room temperature prior to the experiment.

[0275] 4) Isotonic saline (Thermo Scientific, Lot #994448) at room temperature.

[0276] Method:

[0277] 1) Four canisters were randomly selected from the experimental and control batches, respectively.

[0278] 2) Bovine whole blood (containing sodium citrate) purchased from a commercial supplier was mixed with isotonic saline to concentrations of 20% and 40% v / v blood at room temperature, respectively. The total volume of the 20% blood mixture was 1000 ml and the total volume of the 40% blood mixture was 500 ml.

[0279] 3) Two jars per test: one experimental vs. one control. The mixed blood and saline solution was introduced into the jars by vacuum suction.

[0280] 4) The volume of red blood cell sedimentation was recorded every minute for 20 minutes according to the existing scale on the jars.

[0281] 5) After 20 minutes, the two sets of images were compared.

[0282] 6) The data was plotted as a function of RBC sedimentation volume vs. time for comparison.

[0283] Results: Figure 16A and 16B The sedimentation of RBCs after introducing the blood saline mixture into control and experimental (heat-treated) jars was compared. Tests were performed using 1000 ml of a 20% blood and saline mixture ( Figure 16A ) and 500 ml of a 40% blood and saline mixture ( Figure 16B ). Both contained 200 ml of bovine blood. Each test was repeated once. The data shows a close overlap of the volume change curves of RBC sedimentation in the 20% blood tests. All RBC volume sedimentations stabilized at about 125 ml after about 15 minutes after introducing the mixture into the control and experimental jars. In the 40% blood tests, although one experimental volume sedimentation lagged, all volume sedimentations stabilized at about 125 ml after 15 minutes. Figure 17 shows images of the sedimented RBCs in the control and experimental jars. Figure 17A (FIGS. I and II) and Figure 17B (FIGS. I and II) The sedimentation of RBCs after introducing the blood and saline mixture into control and experimental (heat-treated) jars was compared. Tests were performed using 1000 ml of a 20% bovine blood and saline mixture ( Figure 17A ) and 500 ml of a 40% blood and saline mixture ( Figure 17B ). Both were known to contain 200 ml of bovine blood. Each test was repeated once.

[0284] The data shows a close overlap of the volume change curves of RBC sedimentation in the 20% blood tests. All RBC volume sedimentations stabilized at about 125 ml after about 15 minutes after introducing the mixture into the control ( Figure 17A ) and experimental ( Figure 17B ) jars.

[0285] In the 40% blood test, the volume of the settled RBCs stabilized at about the 125 ml volume marker after 15 minutes, despite a volume settling lag in one of the experiments. Figure 17A Figure 17B Figure 17A and 17B The volume of the settled RBCs in the control (17A) and experimental (17B) cans is shown.

[0286] The studies demonstrated no discernible difference between the functionality of the control and experimental cans after heat treatment. The polyDADMAC coated cans showed no degradation of the polyDADMAC functionality or reduction in the effectiveness of promoting RBC flocculation in the liquid after six weeks of aging testing at 55°C. It is expected that the flocculant coated cans (polyDADMAC coated cans) will have a shelf life of at least one year without loss of flocculant activity to provide blood volume estimation in fluids stored at room temperature.

[0287] The above examples are intended to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the methods for predicting selected modifications that can be made to a target biomolecule, but are not intended to limit the scope of the disclosure as believed by the inventors. Those of ordinary skill in the art can make modifications to the above-described modes for carrying out the present disclosure, and it is intended to fall within the scope of the following claims.

[0288] It should be understood that the present disclosure is not limited to a particular method or system, which can of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0289] A number of embodiments of the present disclosure have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. Therefore, other embodiments are within the scope of the following claims.

Claims

1. A method for estimating the volume of blood in a fluid for non-diagnostic purposes comprising: providing a collection device for collecting the fluid, wherein the collection device has a known red blood cell fill rate that is specific for the blood; determining the volume of settled red blood cells after a time period for the red blood cells of the fluid to form a stable settled level in the collection device; and determining an amount of blood in the fluid, wherein the amount of blood in the fluid is a volume V calculated according to the formula b : V b = V m / (Hct× ) where V m is the stable sedimentation level of red blood cells in the collection device, Hct is the mean hematocrit, and is the red blood cell fill rate of the collection device.

2. The method of claim 1, wherein the collection device comprises a red blood cell flocculating agent, and the red blood cell flocculating agent is provided as a dry weight of flocculating agent or as a flocculating agent amount in a carrier solution or as a pre-coat on the collection device.

3. The method of claim 1, wherein the collection device comprises a red blood cell flocculating agent, and the red blood cell flocculating agent is selected from the group consisting of polyethyleneimine, positively charged polyacrylamide, alum or combinations thereof.

4. The method of any one of claims 2 to 3, wherein the amount of the red blood cell flocculating agent is 0.3 to 0.75 volume / volume % of the volume of the collection device, and the red blood cell flocculating agent is polydiallyldimethylammonium chloride.

5. The method of claim 1, wherein the collection device is selected from the group consisting of a canister or a collection bag.

6. The method of claim 1, wherein the collection device is a container adapted to collect and visually estimate the volume of blood.

7. The method of claim 6, wherein the volume of the container is 100 ml, 200 ml, 250 ml, 300 ml, 400 ml, 500 ml, 1200 ml or 2500 ml.

8. The method of claim 1, wherein the fluid comprises normal saline, surgical aspirate, urine, bile, saliva, digestive fluid, cerebral fluid, lymphatic fluid, peritoneal fluid, amniotic fluid or any mixture or combination thereof.

9. Fluid collection device for estimating the amount of blood in a fluid, comprising a blood indicating panel, said panel comprising a series of calibration marks corresponding to approximate amounts of blood V in said fluid, wherein each calibration mark is determined according to the formula: V = 0.5 * (L - L0) / (L0 - L1) where L is the length of the calibration mark, L0 is the length of the first calibration mark and L1 is the length of the last calibration mark. b a corresponding series of calibration marks, wherein each calibration mark is determined according to the formula: V b = V m / (Hct× ) where V b is the volume of blood, V m is the volume of settled red blood cells, Hct is hematocrit, and is the red blood cell fill rate of red blood cells specific to the blood in the collection device.

10. The fluid collection device of claim 9, wherein each of the calibration marks provides an approximation of the volume of blood Vb in the fluid in the collection device, and is calculated from the volume measurement Vm of the red blood cells settled from the fluid within the collection device, a determined red blood cell fill rate of the red blood cells within the collection device and an average hematocrit value.

11. The fluid collection device of claim 9, comprising a red blood cell flocculating agent.

12. The fluid collection device of claim 11, wherein the red blood cell flocculating agent is provided as a dry weight of flocculating agent or as a flocculating agent amount in a carrier solution or as a pre-coat on the fluid collection device.

13. The fluid collection device of claim 11, wherein the red blood cell flocculating agent is selected from the group consisting of polydiallyldimethylammonium chloride, polyethyleneimine, positively charged polyacrylamide, alum or combinations thereof.

14. The fluid collection device of any one of claims 11 to 13, wherein the amount of the red blood cell flocculating agent is 0.3 to 0.75 volume / volume % of the volume of the fluid collection device, and the red blood cell flocculating agent is polydiallyldimethylammonium chloride.

15. The fluid collection device of claim 9, wherein the fluid collection device is selected from the group consisting of a canister or a collection bag.

16. The fluid collection device of claim 9, wherein the fluid collection device is a container adapted to collect and visually estimate the volume of blood.

17. The fluid collection device of claim 16, wherein the volume of the container is 100 ml, 200 ml, 250 ml, 300 ml, 400 ml, 500 ml, 1200 ml, or 2500 ml.

18. The fluid collection device of claim 9, wherein each mark in the series of calibration marks corresponds to a volume of blood contained in the fluid collection device.

19. A system for estimating the volume of blood in a fluid, the system providing for the practice of a method comprising: providing a collection device for collecting the fluid, wherein the collection device has a known red blood cell fill rate that is specific for the blood; determining the volume of settled red blood cells after a time period for the red blood cells of the fluid to form a stable settled level in the collection device; and determining an amount of blood in the fluid, wherein the amount of blood in the fluid is a volume V calculated according to the formula b : V b = V m / (Hct× ) where V m is the stable sedimentation level of red blood cells in the collection device, Hct is the mean hematocrit, and is the red blood cell fill rate of the collection device.

20. The system of claim 19, wherein the collection device comprises a red blood cell flocculating agent.

21. The system of claim 20, wherein the red blood cell flocculating agent is provided as a dry weight of flocculating agent or as a flocculating agent amount in a carrier solution or as a pre-coat on the collection device.

22. The system of claim 20, wherein the red blood cell flocculating agent is selected from the group consisting of poly diallyl dimethyl ammonium chloride, polyethylene imine, positively charged polyacrylamide, alum or a combination thereof.

23. The device of any one of claims 20 to 22, wherein the amount of red blood cell flocculating agent is 0.3 volume / volume % to 0.75 volume / volume % of the volume of the collection device and the red blood cell flocculating agent is poly diallyl dimethyl ammonium chloride.

24. The system of claim 19, wherein the collection device is selected from the group consisting of a canister or a collection bag.

25. The system of claim 19, wherein the collection device is a container adapted to collect and visually estimate the volume of blood.

26. The system of claim 25, wherein the volume of the container is 100 ml, 200 ml, 250 ml, 300 ml, 400 ml, 500 ml, 1200 ml, or 2500 ml.

27. The system of claim 19, wherein the fluid comprises normal saline, surgical aspirate, urine, bile, saliva, digestive fluid, cerebral fluid, lymphatic fluid, peritoneal fluid, amniotic fluid or any mixture or combination thereof.

28. The system of claim 20, wherein the time period for the red blood cells of the fluid to form a stable settled level in the collection device is 15 minutes at room temperature.

29. The system of claim 20, wherein the time period for the red blood cells of the fluid to form a stable settled level in the collection device is 10 minutes at room temperature.

30. The system of claim 19 or 20, wherein the time period for the red blood cells to form a stable settled level is a change in the volume of settled red blood cells of less than 0.5% per minute.

31. The system of claim 19, further comprising a plumbing system.

32. The system of claim 31, wherein the tubing system comprises a first length of tubing adapted to draw fluid through the first port onto the fluid collection device, and a second length of tubing adapted to connect to a source of suction and the second port of the fluid collection device.

33. A kit for estimating the amount of blood in a fluid comprising the fluid collection device of any one of claims 9 to 18 and a tubing system.

34. The kit of claim 33, wherein the tubing system comprises a first length of tubing adapted to draw fluid through the first port onto the fluid collection device, and a second length of tubing adapted to connect to a source of suction and the second port of the fluid collection device.

35. The kit of claim 33 or 34, wherein the kit further comprises instructions.

36. A blood measurement indicator panel comprising a panel material having two sides, a front side and a back side, the front side having a first series of markings and a second series of markings, the first series of markings providing a measure of the volume of settled red blood cells, and the second series of markings providing a measure of the volume of blood, wherein the second series of markings for the volume of blood is determined by the following formula: V b = V m / (Hct× ) where V b is the volume of blood in milliliters, V m is the volume of settled red blood cells in milliliters, Hct is the mean corpuscular hematocrit value, and is the packing fraction of settled red blood cells.

37. The blood measurement indicator panel of claim 36, wherein the first series of markings provides a measure of the volume of settled red blood cells in a collection container.

38. The blood measurement indicator panel of claim 37, wherein the settled red blood cells are human flocculated red blood cells.

39. A blood measurement indicator panel comprising a panel material having a front side and a back side, the front side comprising a first series of volume markings and a second series of volume markings, the first series of volume markings corresponding to the volume of settled red blood cells, and the second series of volume markings corresponding to the volume of blood, wherein the second series of volume markings is determined by the following formula: V b = V m / (Hct× ) where V b is the volume of blood in milliliters, V m is the volume of settled red blood cells, Hct is the mean corpuscular hematocrit value, and is the packing fraction of settled red blood cells.

40. The blood measurement indicator panel of claim 36 or 39, wherein the panel comprises a volume calibration marking for placing the panel on the surface of a collection container.

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