HK1231958B - Rapid measurement of formed blood component sedimentation rate from small sample volumes - Google Patents

Description

Rapid measurement of sedimentation rates of formed blood components from small sample volumes

Background of the Invention

The erythrocyte sedimentation rate (ESR), also known as sedimentation rate or Biernacki reaction, is the rate at which red blood cells sediment, usually measured over a period of one (1) hour. It is a common hematology test and a nonspecific measure of inflammation. To perform the test using traditional techniques, anticoagulated blood is placed in an upright tube, known as a Westergren-Katz tube, and the rate at which the red blood cells sediment is measured and recorded in mm/hour. Specifically, the Westergren method requires the collection of 2 ml of venous blood into a tube containing 0.5 ml of sodium citrate. The sample should be stored at room temperature for no more than 2 hours or at 4°C for no more than 6 hours. Blood is drawn into the Westergren-Katz tube to the 200 mm mark. The tube is placed in an absolutely vertical position in a rack at room temperature for one hour, at which time the distance from the lowest point of the surface meniscus to the interface between the red cell-free plasma and the portion of the sample occupied by red cells is measured. The distance the red blood cell interface moves expressed in millimeters per hour (mm/h) is the ESR.

The ESR is governed by a balance between pro-sedimentation factors, primarily fibrinogen (but may also refer to levels of serum C-reactive protein (CRP), immunoglobulins A and G, α(1)-acid glycoprotein, and α(1)-antitrypsin), and anti-sedimentation factors, primarily the negative charge of the red blood cells (zeta potential). In one example of the effects of inflammation, high concentrations of fibrinogen in the plasma cause the red blood cells to adhere to each other. The red blood cells adhere to form stacks known as "rouleaux," which sediment faster than individual red blood cells. Rouleaux formation may also occur in association with lymphoproliferative disorders in which one or more immunoglobulins are found in high concentrations. However, rouleaux formation may be a normal physiological finding in horses, cats, and pigs.

The ESR increases with any cause or focus of inflammation. It increases during pregnancy and rheumatoid arthritis, while it decreases in polycythemia vera, sickle cell anemia, hereditary spherocytosis, and congestive heart failure. The baseline ESR is slightly higher in women.

The standard method for measuring ESR is the Westergren test, which uses a large volume of blood, typically several ml. Since many samples have an ESR as low as 10 mm/hour, it typically requires an hour of incubation. Inflammatory factors that increase the ESR include fibrinogen, C-reactive protein (CRP), and some immunoglobulins, which can increase the ESR to as high as 100 mm/hour.

Conventional techniques for performing sedimentation tests have various limitations. For example, as discussed, the Westergren sedimentation test requires the drawing of a relatively large volume of blood. Furthermore, conventional sedimentation testing techniques take a significant time period and can result in time lags in obtaining test results. This time lag can lead to delays in diagnosis and treatment, which can have a detrimental impact on a patient's health.

Incorporation by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Summary of the Invention

It may be desirable to have a sedimentation rate test that can be completed in the very short time, such as, but not limited to, completing on the magnitude of a few seconds to a few minutes. For distributed inspection arrangements, it may also be desirable to have a sedimentation rate measurement that only uses a small blood volume, such as can be obtained by alternative positions, non-venous blood sampling or minimum venous blood sampling. It may also be desirable to carry out sedimentation measurement and create an objective record of the measurement in an automated manner (without the need for manual observation). In addition, it is possible to obtain further information useful in the management of optimizing patients by performing and/or maximizing the speed of the multiplexed measurement of other analytical parameters parallel to the sedimentation rate measurement.

In one embodiment described herein, the sedimentation rate measurement method can utilize (1) centrifugation techniques for separating red blood cells from plasma and (2) video and/or static imaging capabilities. Both can be used alone or in combination to accelerate the sedimentation of red blood cells and measure their rate. Of course, techniques for accelerating sedimentation other than centrifugation can be used in place of or in combination with centrifugation to separate blood components.

In one non-limiting example, the method can advantageously (1) enable rapid measurement (in seconds) of the ESR with small blood sample volumes, such as about 20-25 microliters ("uL" or "μL") or less, (2) enable determination of both the red blood cell sedimentation rate and the hematocrit using automated image analysis, and/or (3) enable automated techniques to compensate for the effect of hematocrit on the uncorrected ESR so as to provide a value corresponding to the traditional Westergren method. Of course, alternative embodiments using larger volumes of blood are not excluded. Due to the ability to correct for hematocrit, some embodiments of the sedimentation measurement techniques described herein are more robust than the traditional Westergren technique and can be used on samples having fibrinogen and/or hematocrit levels outside the narrow range required by the Westergren test.

Using the embodiments herein, a corrected ESR can be obtained in about a few seconds using a small blood volume and which compensates for the effect of hematocrit on the ESR. Obtaining results in about a few seconds during the initial centrifugation can accelerate the delivery of diagnostics to patients.

Furthermore, in the context of multiplexed assay procedures, common pre-treatment steps already involve separating red blood cells and white blood cells from plasma or serum prior to measurement of cell markers and analytes present in plasma/serum. Thus, it is convenient to combine ESR measurements with such pre-treatment procedures, which will have been performed during the assay preparation process. The ESR measurement will not impose a significant burden on the use of limited amounts of blood available from non-venous collection methods, either in terms of additional processing time or non-venous collection methods. By way of non-limiting example, it will be understood that the assay process, including (one or more) pre-treatment steps, can occur in a single instrumented system. Alternatively, some embodiments may perform one or more steps in one instrument and another one or more steps in another instrument.

It should also be understood that the embodiments described herein may be adapted to have one or more of the features described below. In one non-limiting example, a typical protocol may involve placing 20 μL of blood in a centrifuge vessel and spinning a pendulum centrifuge rotor at 4000 rpm (580 g) for approximately 10 seconds. During this period, the interface between the sample portion containing red blood cells and the sample portion cleared of red blood cells is observed using video imaging. While other time periods are not excluded, obtaining ESR measurements within this short time period may be advantageous. Optionally, some embodiments may correct these "raw" ESR values for the effects of hematocrit. Hematocrit may be measured in the same operation as that used to measure the raw ESR. In one non-limiting example, after a relatively low-speed rotation to measure the ESR during centrifugation, the rotation speed is increased to pack the red blood cells. The hematocrit is determined by image analysis of the packed red blood cells and the volume of supernatant plasma. Alternatively, other techniques for measuring hematocrit may be used to correct the "raw" ESR values.

At least one of the embodiments herein can correct for ESR without using calculation of the slope of a substantially linear transformation of the non-linear (exponential) portion of the sedimentation curve.

At least one of the embodiments herein can correct the ESR without calculating a mathematical function of multiple red blood cell/plasma interface locations that occur in the nonlinear portion of the sedimentation curve.

At least one of the embodiments herein may correct the ESR without selecting a section of the sedimentation curve that is located in the non-linear portion of the sedimentation curve.

At least one of the embodiments herein may correct the ESR based solely on measurements of the linear portion(s) of the sedimentation curve.

At least one of the embodiments herein may calibrate the ESR based on a measurement consisting essentially of the linear portion(s) of the sedimentation curve. By "consisting essentially of," it is meant that at least 90% or more of the measurement is based on the linear portion(s).

At least one of the embodiments herein may correct the ESR without determining a mathematical function of a nonlinear segment of a sedimentation curve that represents the magnitude of erythrocyte repulsion between cells in a blood sample.

At least one of the embodiments herein can correct for ESR without negating the time period during centrifugation of the sample that forms the linear portion of the sedimentation curve.

At least one of the embodiments herein may correct the ESR for hematocrit using hematocrit measurements other than those obtained from centrifugation techniques, such as, for example, lysis of red blood cells with detergent and mixing with ferrocyanide and cyanide, followed by measurement of the absorbance of the resulting cyanomethemoglobin.

At least one of the embodiments herein may adjust a blood sample so that it is at a known hematocrit level for sedimentation measurement.

In at least one embodiment described herein, a method is provided, comprising: applying an accelerated blood component separation technique to a blood sample for a period of time to separate formed blood components from plasma; determining a sedimentation rate of the formed blood components based at least on: a time-dependent compaction curve and a hematocrit correction factor, wherein a time-dependent compaction curve is established for at least one formed blood component in the blood sample after the accelerated blood component separation has begun, the compaction curve having an initial approximately linear portion and a nonlinear portion following the linear portion.

In at least one embodiment described herein, a method is provided, comprising: centrifuging a blood sample in a vessel for a period of time; establishing a time-dependent compaction curve for at least one formed blood component in the blood sample after centrifugation has begun, the compaction curve having an initial approximately linear portion; and correcting the effect of hematocrit on the sedimentation rate of the formed blood component by applying a hematocrit correction factor to the approximately linear portion of the compaction curve.

It should be understood that the embodiments in the present disclosure may be adapted to have one or more features described below. In one non-limiting example, the method includes calibrating the sedimentation rate according to a centrifuge-based technology with a sedimentation rate according to a reference technology. Optionally, the reference technology is the Westergren technology. Optionally, the sample is about 25uL less. Optionally, centrifugation occurs at a first speed for a first time period, followed by a second, faster speed for a second time period. Optionally, centrifugation includes using a centrifuge configured to allow visual observation of the blood sample during centrifugation to establish the interface position of one or more formed blood components in the blood sample. Optionally, centrifugation includes using a centrifuge with a window thereon to enable visual observation of the blood sample, thereby establishing the red blood cell/plasma interface position over time. Optionally, centrifugation includes using a centrifuge, a light source, and an image capture device to enable visual observation of the blood sample, thereby establishing the formed blood component/plasma interface position over time. Optionally, compaction curve data is collected by capturing multiple images of the interface locations of one or more formed blood components in the centrifuge vessel over the time period. Optionally, the pixel positions in the multiple images are used to accurately determine the interface locations. Optionally, compaction curve data is collected by capturing a single image of the interface locations of one or more formed blood components in the centrifuge vessel after a period of time, wherein the sedimentation rate is calculated based on the position of the meniscus of the supernatant liquid and the interface location. Optionally, the compaction curve data is collected while the sample is undergoing centrifugation. Optionally, centrifugation is used to obtain a hematocrit measurement and used to correct the effect of hematocrit on the sedimentation rate measurement. Optionally, correcting the hematocrit includes calculating a mathematical function of the multiple formed blood component interface locations present in the curve, wherein the function can be used to correct for sedimentation rate variations due to hematocrit. Optionally, the hematocrit measurement in the sample is obtained using a technique separate from centrifugation. Optionally, an image transformation is used to convert a curved interface to a flat interface. Optionally, image transformation parameters are selected such that a video of the interface locations of the formed blood components is subjected to an image transformation, thereby selecting a region of interest that covers the entire range of positions of both the air/plasma interface and the red blood cell interface. Optionally, for each time point in the video, the pixel intensity values within the region of interest across each row of the sample vessel containing the sample are averaged to produce a single column representing the intensity radially downward along the sample vessel. Optionally, the linear region of the sedimentation profile is used to extract the sedimentation rate. Optionally, the formed blood components are white blood cells. Optionally, the formed blood components are platelets.

It should be understood that embodiments of the present disclosure may be adapted to have one or more features described below. In one non-limiting example, the method includes performing an image transformation on the image to transform the image having a curved interface into a corrected image having a straight linear interface; establishing a time-dependent compaction curve for at least one formed blood component in the blood sample based on the interface position in the corrected image after centrifugation has begun. Optionally, the method includes: transferring at least a portion of the blood sample from a blood sample position to a centrifuge vessel using a system controlled by a programmable processor; transferring the vessel from a first addressable position to a centrifuge having a second addressable position using a sample handling system controlled by a programmable processor; centrifuging the blood sample in the vessel for a period of time; collecting at least one image of the interface position of the formed blood component and plasma after centrifugation; establishing a time-dependent compaction curve for at least one formed blood component in the blood sample based on the interface position (one or more) in the corrected image after centrifugation has begun. Optionally, removing the vessel from the centrifuge to obtain the image. Optionally, returning the vessel to the centrifuge after obtaining the image. Optionally, the method includes varying the centrifugation speed to establish a linear compaction profile for at least one formed blood component over the time period until compaction is complete; monitoring the centrifugation speed profile for at least a portion of the time period; and determining a sedimentation rate of the blood component based on the centrifugation speed profile. Optionally, the method includes collecting at least a first single image of the position of the interface between the formed blood component and plasma at an initial time; collecting at least a second single image of the position of the interface between the formed blood component and plasma at a second time when the rate of sedimentation is still linear; and calculating the sedimentation rate of the at least one formed blood component in the blood sample based on the calculated linear sedimentation rate and a hematocrit correction factor.

In another embodiment described herein, a method is provided, comprising: centrifuging a blood sample in a vessel for a period of time; establishing a sedimentation rate using an image of the vessel under a single state condition; and correcting for the effect of the hematocrit on the sedimentation rate of formed blood components by using a hematocrit correction factor. As used herein, using imaging may include determining the sedimentation rate using a single image of the vessel. By way of non-limiting example, when using a single image of the vessel, the meniscus of the supernatant liquid in the vessel indicates an initial level and the position of the interface of the formed components with the supernatant liquid indicates a current position, from which the sedimentation rate is calculated. Optionally, some embodiments may use multiple images for sedimentation rate calculation, but all images are images of the vessel when the vessel is under a single state condition. Optionally, all images are images of the vessel at a single point in time. Optionally, all images are images of the vessel when the position of the interface of the formed components in the vessel does not change. In one non-limiting example, dual speed is used to extend the dynamic range of the ESR measurement. Alternatively, some embodiments may use centrifugation at three or more speeds rather than just two. Although described herein in the context of a centrifuge, it should be understood that the centrifuge speeds referred to herein can be viewed as a proxy for the G forces applied to the sample, and that the concepts herein can be applied to other embodiments that provide accelerated sedimentation of formed sample fractions but do not use a centrifuge.

In another embodiment described herein, a method is described, the method comprising using an accelerated blood component separation technique on a blood sample in a vessel for at least a period of time; capturing at least a first single image of the position of the interface between formed blood components and plasma in the vessel at an initial time; capturing at least a second single image of the position of the interface between formed blood components and plasma at a second time within a linear sedimentation time period; and calculating a sedimentation rate of at least one formed blood component in the blood sample based on the calculated linear sedimentation rate and a hematocrit correction factor.

It should be understood that embodiments of the present disclosure may be adapted to include one or more of the features described below. In one non-limiting example, the accelerated blood component separation technique includes centrifugation. Optionally, the method further includes calibrating the sedimentation rate according to the centrifuge-based technique with the sedimentation rate according to a reference technique. Optionally, the reference technique is an automated Westergren technique. Optionally, the reference technique is a manual Westergren technique. Optionally, the blood sample is approximately 25 μL or less. Optionally, centrifugation occurs at a first speed for a first period of time, followed by a second, faster speed for a second period of time. Optionally, the first speed is configured to apply a substantially uniform force to the sample, while the second speed is configured to apply a force greater than the substantially uniform force associated with the first speed, but within a wider force variation range. Optionally, the accelerated blood component separation technique occurs at a first G-force for a first period of time, followed by a second, higher G-force for a second period of time. Optionally, the first G-force applies a substantially uniform force to the sample, while the second first G-force applies a substantially uniform force greater than the substantially uniform force associated with the first G-force, but within a wider force variation range. Alternatively, the first G-force is in the range of about 35G to about 45G. Alternatively, the first G-force is in the range of about 30G to about 50G. Alternatively, the first G-force is in the range of about 10G to about 60G. Alternatively, the second G-force is in the range of about 10G to about 100G. Alternatively, the first G-force is sufficient to accelerate sedimentation but not so fast that the formed component becomes fully compacted before a sedimentation change is observed when the sedimentation change is in the linear range. Alternatively, the second G-force has a high speed rotation that is at least about 2 times or more of the measured rotation at a lower speed.

[0011] In yet another embodiment described herein, a method is described that includes applying a force to a blood sample to reduce a measurement time associated with calculating a sedimentation rate of at least one formed blood component in the blood sample.

In another embodiment described herein, a device for use with a sample is provided, comprising a centrifuge having a centrifuge vessel holder configured to allow detection of the interface position of blood components in the vessel holder during centrifugation. Optionally, the centrifuge has a window to allow visual observation of the centrifuge vessel holder during centrifugation. Optionally, the centrifuge has an illumination source to allow detection of the interface position of blood components in the sample.

In another embodiment described herein, a system is provided comprising: a centrifuge having a centrifuge vessel holder configured to allow detection of an interface position of blood components in the vessel holder during centrifugation; a sample handling system for transporting a blood sample from a first location to a location on the centrifuge; and a processor programmed to record the interface position during at least a portion of the centrifugation.

It should be understood that the embodiments in the present disclosure include a method comprising at least one technical feature from any other embodiment herein. Alternatively, a method may include at least any two technical features from any other embodiment herein.

Alternatively, a device may include at least one technical feature from any other embodiment herein. Alternatively, a device may include at least any two technical features from any other embodiment herein. Alternatively, a system may include at least one technical feature from any other embodiment herein. Alternatively, a system may include at least any two technical features from any other embodiment herein.

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a graph of erythrocyte sedimentation of high, medium, and low ESR blood samples in Westergren-Katz tubes.

2A-2B are images of blood samples in a transparent centrifuge vessel.

FIG3 shows a schematic diagram of a centrifuge with an embodiment of a detection system.

4-5 illustrate images captured using one embodiment of a detection system.

6A-7C show a series of rectified and unrectified images of an interface in a blood sample subjected to centrifugation.

8A-8B show various kymograms for one test sample.

FIG9 shows a graph of the sedimentation curve for one test sample.

10A-10B show graphs of sedimentation curves fitted to the data plotted on FIG. 9 using various fitting functions.

11-14 are graphs showing various sample sedimentation characteristics for samples having various levels of added fibrinogen.

FIG15 shows the sedimentation rates of several blood samples manipulated to have different hematocrit levels.

16 is a graph of interface position over time for samples having different hematocrit levels also shown in FIG. 15 .

FIG. 17 is a graph of the interface position over a 10 second period for samples having varying hematocrit levels.

FIG18A shows a graph of the ESR curve for an embodiment of the present disclosure without hematocrit correction.

FIG18B shows a graph of the ESR curve for an embodiment herein that has been corrected for hematocrit.

18C shows a graph of hematocrit measurement based on hemoglobin concentration, according to one embodiment herein.

19 and 20 illustrate the sedimentation rates of several samples (as detailed in FIG. 15 ) plotted using non-LOG and LOG axes.

FIG21 shows a kymogram illustrating the white blood cell interface.

22 shows a schematic diagram of one embodiment of an integrated system having a sample processing component, a pre-processing component, and an analysis component.

DETAILED DESCRIPTION

It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and do not limit the invention as claimed. It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a material" may include a mixture of multiple materials, reference to "a compound" may include multiple compounds, and so on. The references cited herein are hereby incorporated by reference in their entirety, except to the extent that they conflict with the teachings expressly set forth in this specification.

Throughout this specification and in the claims that follow, reference will be made to several terms which shall be defined to have the following meanings:

"Optional" or "optionally" means that the subsequently described event may or may not occur, and thus, the description includes instances where the event occurs and instances where the event does not occur. For example, if a device optionally includes a feature for a sample collection well, this means that the sample collection well may or may not be present, and thus, the description includes both configurations in which the device includes the sample collection well and configurations in which the sample collection well is not present.

Referring now to Figure 1, the history of the red blood cell/plasma interface of a group of blood samples is shown. Figure 1 shows the history of red blood cell sedimentation in a Westergren-Katz tube for a series of samples, wherein the solid line shows high ESR, the dotted line shows medium ESR, and the dot shows low ESR. Although the Westergren ESR is recorded as a single number (mm/hour) as seen in Figure 1, the sedimentation rate varies significantly over an hour, starting slowly, increasing, and then decreasing. The standard Westergren method records the ESR at a single location over an hour to give the average sedimentation rate over that hour. A newer method called Sigma ESR has been shown to correlate better with clinically relevant variables by taking the sum of the distances moved at 20, 30, 40, 50, and 60 minutes.

Sedimentation curve measurement

A variety of techniques can be used to establish a sedimentation rate curve for one or more formed blood components. Although the present application is primarily described in the context of measuring the sedimentation rate of red blood cells, the systems and methods herein can also be adapted for use in measuring the sedimentation rates of other formed blood components, such as, but not limited to, measuring the sedimentation rates of white blood cells, platelets, etc.

In a non-limiting example, a kind of technology described herein comprises taking images at several time points during sedimentation by the following: the sample vessel is placed in a centrifuge, rotates for a few seconds, stops rotating, removes the vessel, is placed in a viewer, takes images and repeats the above-mentioned items to obtain multiple images over time. From the perspective of device simplicity, it is helpful in that it simplifies the hardware implementation for obtaining such images. Other places herein discuss the ability to measure sedimentation, wherein the slope of the initial (linear) part of the sedimentation curve is used to calculate ESR.

Of course, it should be understood that some embodiments can obtain such images/data related to the position of the interface while the container is in situ in the centrifuge, without having to stop the centrifuge to remove the sample vessel for imaging. In situ images can be captured while the centrifuge rotor is in motion or at rest. It should also be understood that while discrete images can be captured, video, continuous imaging, and multiple frames per second imaging can also be used.

2A and 2B, there is shown an example of the red blood cell interface before centrifugation and in the early stages of centrifugation. For non-limiting example, the centrifugal vessel can be made of transparent materials such as transparent plastics (injection molded polystyrene) in whole or in part. In some embodiments, the transparent portion can be a window, a translucent port or a vessel that is aligned with a translucent strip for allowing the desired blood component interface of the sample in the vessel to be imaged. In the present embodiment, the radius of the centrifugal vessel at its midpoint is 35mm (radial distance from the axis of rotation). In one embodiment, the outer radius is 35mm, and the inner radius (that is, to the upper surface of the liquid) is 28mm, so that the midpoint is 31.5mm. The sample length in the vessel is 7mm and the vessel inner diameter is 2.3mm. The variation of the sample vessel geometry or the sample volume to be tested can be illustrated by recalibrating the empirical parameters for the hematocrit correction factor as discussed elsewhere herein.

Other suitable centrifuge designs and features, including the dimensions of the centrifuge vessel, the configuration of the centrifuge rotor, and the size of the centrifuge, are disclosed in co-pending U.S. patent applications Serial Nos. 13/355,458 and 13/244,947, all of which are hereby incorporated by reference in their entirety for all purposes. Other components of the system, including suitable imaging devices and fluid handling systems, are also described in the applications incorporated by reference. For example, the capabilities of digital cameras, such as those described in those applications, can be used to measure very small distances and rates of change of distances to measure ESR. Image analysis can be used to measure the movement of the interface between red blood cells and plasma.

For non-limiting example, in some embodiments, only two measurements are required in the early stage (within a few seconds) after centrifugation has begun to clearly determine the sedimentation rate with high accuracy. In one embodiment, a first image can be taken after reaching the initial minimum centrifuge speed, and then a second image can be taken after about 10 seconds. Of course, images can be taken at other time periods as long as they are in the linear portion of the sedimentation curve.

Looking at Figures 2A and 2B, the position of the RBC interface in a stationary (vertical) tube (short-term centrifugation) can be seen. In this non-limiting example, the swing-out centrifuge vessel is stopped and oriented vertically in Figures 2A and 2B. Of course, since surface tension holds the interface in place, stationary imaging does not require the tube to be vertical (as long as stationary imaging is completed quickly). Typically, this is accomplished within one or two seconds, otherwise the RBC interface will begin to flow.

As seen in Figures 2A and 2B, there is a sharp transition clearly visible between the part occupied by red blood cells and plasma of the sample. The red blood cell interface liquid level at the level is clearly visible in these images. In Figure 2B, the distance that this interface moves relative to Figure 2A corresponds to a large amount of pixels (50/mm) in the image. Therefore, as seen, the number of pixels that the interface travels allows the accurate tracking of the interface position change. Of course, other image resolutions such as but not limited to 50 pixels/mm to 1000 pixels/mm (or higher) can be used to provide larger granularity with respect to the number of pixels per mm or every other unit length. As long as the resolution is enough to accurately determine the change of the interface position, pixels less per unit length can be used. Some embodiments can amplify the image so that more pixels are associated with the interface and thus more pixels are associated with the position change of the interface. Some embodiments can use a detector with a larger number of pixels per unit area. This improves the sensitivity of measurement by measuring more pixels and having the ability to detect even more subtle changes in the interface position.

In one embodiment, a method is provided, which uses a transparent window in the centrifuge housing so that sedimentation can be video recorded during low-speed centrifugation. Furthermore, the centrifugal field causes the meniscus to become straighter (at right angles to the centrifugal force vector), thereby making it easier to measure the small sedimentation distance. This may be particularly correct when capturing images while the centrifuge rotor is rotating. By rotating a small volume (20-25uL) of blood at an intermediate speed (typically 4000rpm, but 1000 to 6000rpm may also be suitable), the almost complete sedimentation of red blood cells was completed in about three minutes in this embodiment. In fact, a method can measure sedimentation for a few seconds at a relatively low speed (1000 to 4000rpm), then increase the speed to about 10000rpm for about three minutes to pack red blood cells and determine the hematocrit. In one embodiment, the low-speed rotation is about 40G. Alternatively, the rotation of some embodiments can create a G force within the range of about 40G to 100G. Alternatively, the rotation of some embodiments may create a G-force in the range of about 10 G to 60 G. Alternatively, the rotation of some embodiments may create a G-force in the range of about 10 G to 100 G. In one embodiment, the desired G-rate is a G-rate that is sufficient to accelerate sedimentation but not so fast that the formed component becomes fully compacted before a change in sedimentation is observed while the change in sedimentation is in the linear range.

In one embodiment, this multi-stage rotation under different centrifugal forces (which are generally linearly related to the centrifuge speed) allows imaging of sedimentation, followed by rapid rotation slowing down to achieve compaction of blood components and separation from plasma. Optionally, some embodiments can have a low-speed rotation within the range of 1000rpm+/-20%rpm. Alternatively, some embodiments can have a low-speed rotation within the range of 800rpm to 1500rpm. Alternatively, some embodiments can have a high-speed rotation that is about 2 times or more of the measured rotation at a lower speed. Alternatively, some embodiments can have a high-speed rotation that is about 3 times or more of the measured rotation at a lower speed. Alternatively, some embodiments can have a high-speed rotation that is about 4 times or more of the measured rotation at a lower speed. Alternatively, some embodiments can have a high-speed rotation that is about 5 times or more of the measured rotation at a lower speed. Alternatively, some embodiments can have a high-speed rotation that is about 6 times or more of the measured rotation at a lower speed. Alternatively, some embodiments can have a high-speed rotation that is about 7 times or more of the measured rotation at a lower speed. Alternatively, some embodiments may have a high speed rotation of about 8 times or more than the lower speed measurement rotation. Alternatively, some embodiments may have a high speed rotation of about 9 times or more than the lower speed measurement rotation. Alternatively, some embodiments may have a high speed rotation of about 10 times or more than the lower speed measurement rotation. Alternatively, some embodiments may have a high speed rotation of about 15 times or more than the lower speed measurement rotation. Alternatively, some embodiments may have a high speed rotation of about 20 times or more than the lower speed measurement rotation.

Alternatively, at low speed, the rotating part is measured, and once the desired RPM is selected from the various ranges previously set forth, a controller or other device can be used to maintain the desired rpm so that the desired forming component measurement can be performed. This desired rpm can be at a controlled rate +/-1% of the target RPM. Alternatively, the controlled rate can be +/-2% of the target RPM. Alternatively, the controlled rate can be +/-3% of the target RPM. Alternatively, the controlled rate can be +/-4% of the target RPM. Alternatively, the controlled rate can be +/-5% of the target RPM. In an embodiment, the process may involve a low-speed centrifuge rotation at a controlled rate, followed by a high-speed rotation, wherein the main factor is to rotate the centrifuge disc above a minimum threshold, wherein the appropriate RPM is less important than maintaining at least the minimum rotation rate.

With reference now to Fig. 3, an embodiment of the centrifuge 100 that can monitor interface position will now be described.In order to monitor sedimentation, image capture device 110 can be positioned near centrifuge 100, along with light source 112 such as but not limited to green LED being positioned to provide illumination from relative position.Alternatively, the illumination light source of another color or another wavelength is not excluded.Image capture device 110 can be a static camera, a high-speed camera, a video camera or other devices that are enough to detect the position of the interface.Of course, other detectors are not excluded, such as but not limited to non-image capture devices. For non-limiting example, a non-visual imaging device here can be a photodiode, which can play the role of a detector to detect when the blood component interface passes through the detector or by a large amount of transmission of light to detect the ratio of the volume blocked by RBC or other (one or more) blood components.Non-visual imaging detectors can be used, even if they may not actually transmit visual images but can still detect the interface liquid level position and/or position change in the sample.

Any description of a camera or other detection device described elsewhere herein may apply. In one example, image capture device 110 may be a digital camera. The image capture device may also include a charge-coupled device (CCD) or a photomultiplier tube and phototube, or a light detector or other detection device such as a scanning microscope (whether back-illuminated or front-illuminated). In some cases, the camera may use a CCD, a CMOS, a lensless (computational) camera (e.g., a Franken camera), an open-source camera, or may utilize any other visual detection technology known or later developed in the art. The camera may include one or more features that enable the camera to focus while in use or that can capture an image that can be refocused later. In some embodiments, the imaging device may employ 2D imaging, 3D imaging, and/or 4D imaging (incorporating changes over time). The imaging device may capture static images. Static images may be captured at one or more points in time. The imaging device may also capture video and/or dynamic images. Video images may be captured continuously over one or more time periods. Any other description of the imaging device and/or detection unit may also apply, preferably as long as they are capable of detecting changes in the position of the interface.

In one non-limiting example, the light source 112 may be a light emitting diode (LED) (e.g., a gallium arsenide (GaAs) LED, an aluminum gallium arsenide (AlGaAs) LED, a gallium arsenide phosphide (GaAsP) LED, an aluminum gallium indium phosphide (AlGaInP) LED, a gallium (III) phosphide (GaP) LED, an indium gallium nitride (InGaN)/gallium (III) nitride (GaN) LED, or an aluminum gallium phosphide (AlGaP) LED). In another example, the light source may be a laser, for example, a vertical cavity surface emitting laser (VCSEL) or other suitable light emitter, such as an aluminum gallium indium phosphide (InGaAlP) laser, a gallium arsenide phosphide/gallium phosphide (GaAsP/GaP) laser, or an aluminum gallium arsenide/gallium arsenide (GaAIAs/GaAs) laser. Other examples of light sources may include, but are not limited to, electron-excited light sources (e.g., cathodoluminescence, electron-excited luminescence (ESL bulbs), cathode ray tubes (CRT monitors), Nixie tubes), incandescent light sources (e.g., carbon bond lamps, traditional incandescent bulbs, halogen lamps, Groh rods, Nernst lamps), electroluminescent (EL) light sources (e.g., light-emitting diodes, organic light-emitting diodes, polymer light-emitting diodes, solid-state lighting, LED lamps, electroluminescent sheets, electroluminescent wires), gas discharge light sources (e.g., fluorescent lamps, induction lighting, hollow cathode lamps, neon and argon lamps, plasma lamps, xenon flash lamps), or high-intensity discharge light sources (e.g., carbon arc lamps, ceramic discharge metal halide lamps, mercury dielectric arc iodine lamps, mercury vapor lamps, metal halide lamps, sodium vapor lamps, xenon arc lamps). Alternatively, the light source may be a bioluminescent, chemiluminescent, phosphorescent, or fluorescent light source.

3 , the centrifuge vessel 114 containing the blood sample therein can be positioned so as to be between the image capture device 110 and the light source 112 so as to enable visualization of the interface(s) of the formed blood components in the vessel. The centrifuge rotor 116 can be configured with openings, windows, or other areas that allow visualization of the centrifuge vessel 114 during centrifugation. Measuring sedimentation can be used during the centrifuge spin, but it should be understood that measuring sedimentation between or after spins while the centrifuge is at rest is not excluded.

In the present embodiment of Figure 3, the axis of rotation of the centrifuge rotor 116 can be vertical. It should be understood that other axes of rotation, such as horizontal or angled axes of rotation, are not excluded. Some embodiments can have a first orientation during one time period and a second orientation during a second or other time period.

In one non-limiting example, the position of the top and/or bottom of the centrifuge vessel is acquired by imaging as a reference point, and these data are subsequently used to calibrate the liquid and interface levels. FIG4 shows a camera view of a centrifuge vessel 114 in a centrifuge. Illumination from a light source 112 behind the centrifuge vessel 114 allows visualization of the blood sample S and the blood/air interface 120. The direction of rotation is shown by arrow 122.

Referring now to FIG5 , a magnified view of the interface(s) of a blood sample in centrifuge vessel 114 will now be described. FIG5 is an image of red blood cell sedimentation during centrifugation. Air/plasma interface 130 and plasma/red blood cell interface 132 are clearly discernible in this image as sharp lines separating spatial regions of differing contrast. Also shown in the image of FIG5 are space 134 above the air/plasma interface, plasma 136, and light blocked by red blood cells 138.

It should be understood that flash illumination or frame capture synchronized to the rotor position is not excluded, but in this embodiment, it is not required for image capture. In this non-limiting example, for the image of Figure 5, the 200ms exposure (relatively short relative to the rotation time) used for CCD camera image acquisition makes interfaces 130 and 132 clearly visible during the rotation (see Figure 5). This is long compared to the rotation period (i.e., many rotations occur during this time, so the image is blurred). The image is blurred around the rotor axis, making the air/plasma interface and the plasma/erythrocyte interface visible as arcs (although there may be flash effects that are also considered). Although data acquisition does not require frame capture to be synchronized to the rotation, some embodiments may use synchronization. Alternatively, some embodiments without synchronization may result in streaks, which can be compensated by a combination of longer exposure and image processing. Other embodiments may use faster image acquisition techniques to generate images that minimize and/or eliminate blur. Some embodiments may use flash illumination or other techniques to capture images of fast-moving objects, such as images of vessels containing samples during centrifugation.

As can be seen in FIG5 , two regions through which light is transmitted through the centrifuge vessel 114 include the air 134 above the liquid and the plasma between the labeled air/plasma interface 130 and the plasma/red blood cell interface 132. The air/plasma interface 130 itself is visible as an arc. Virtually no light is transmitted through the region of the vessel 114 where the red blood cells are located (although it should be understood that this region is not completely dark due to the light transmitted when the vessel 114 is rotated out of the blocking position).

In one embodiment, with five frames per second, capture images for three minutes with long exposure (～200ms), then process image to extract sedimentation curve. Optionally, the imaging speed includes but is not limited to 1,2,4,8,16,32,64 or 128 images per second. Optionally, exposure time includes but is not limited to 10,20,40,80,160,320 or 640ms. The temperature during measurement may also change. Although many embodiments of this paper are measured at room temperature, other temperatures, for example, 37 ℃ are not excluded. The influence of temperature will be considered in calibration, such as in order to determine the empirical parameters of the correction factor. In addition, the time of centrifuge rotation acceleration is generally about 3 seconds, but faster or slower rotation acceleration time is not excluded.

By way of non-limiting example, the sedimentation rate of the desired formed blood component being measured may be defined by:

1) Fitting the plasma/red blood cell interface position to time exponentially, or

2) Taking the (linear) rate of movement of the interface in the first few seconds to give a parameter that can then be related to the Westergren ESR.

While other arrangements are not excluded, it will be appreciated that the time of sedimentation is typically defined to begin after the rotor 116 reaches a target speed, at which time the buckets holding the centrifuge vessel 114 are oriented radially in the plane of rotation and are therefore in an optimal position for image capture and processing.

Data preprocessing

Image Transformation

6A-6B , one embodiment of the present invention may utilize an image preprocessing step prior to analysis that may be a combination of (1) conversion of the interface arc to a flat interface and (2) rotation of the image to compensate for any minor offsets in the radial direction. This aligns off-center pixels with the central axis in a manner that has negligible effect on the y-position of the interface, so the blurred arcs from the rotated tube are now horizontal streaks.

As seen in Figures 6A-6B, the initial image transformation can be used to compensate for the arc. The image in Figure 6B shows a rectangle 150 with a selected region of interest across which horizontal averaging is performed, and two short horizontal lines showing where the algorithm has identified the locations of the air/plasma interface 130 and the plasma/red blood cell interface 132.

This image transformation is required to remove the effects of the vertical lines seen in FIG6A , which are caused by the shimmer effect between the centrifuge's rotation frequency and the camera's acquisition frequency. Thin vertical (i.e., radial) section measurements will be susceptible to these lines, which slowly move across the image, but the straightening transformation allows averaging in the x-direction (at right angles to the radial direction) and frees the profile from the effects of moving lines. This procedure also improves the signal-to-noise ratio.

Referring now to Figures 7A-7C, examples showing different degrees of arc compensation are shown. A selection of image transformation parameters can be selected to introduce a desired level of correction. Figure 7A shows too little compensation. Figure 7B shows just enough compensation. Figure 7C shows too much arc compensation.

For each data set using a script that generates a series of images with different arc and rotation corrections, superimposing a series of horizontal lines 160 on the image allows for a determination of when the interface is flat (horizontal in the images of Figures 7A-7C). This determination of the appropriate degree of arc correction can be determined by a programmable processor configured for image processing, can be pre-set based on a calibration procedure, or can be selected based on manual inspection.

Once these parameters are selected, the acquired image information, which may be a video, is passed through the transformation. The region of interest can be selected to cover the entire positional range of both the air/plasma interface 130 and the red blood cell interface 132. Alternatively, some embodiments can select a region of interest that covers only one of the interfaces 130 or 132. Alternatively, some embodiments can be configured to target one or more other regions of interest in the sample.

Sedimentation curve extraction

8A-8B , for each time point in a plurality of images, one embodiment of the present technology averages the pixel intensity values for each row (across the vessel 114) within the region of interest 150 to produce a single column representing the intensity radially down the vessel. The columns for each time point are then assembled into a kymogram, i.e., an image in which the x-axis represents time and the y-axis represents radial position along the vessel.

FIG8A shows a kymogram according to one embodiment described herein. The kymogram of FIG8A shows the average image intensity along the tube (y-axis) over time (x-axis). FIG8A shows the air interface 130, plasma 136, plasma/red blood cell interface 132, and red blood cells 140. More specifically, the dark horizontal line near the top of the image represents the air/plasma interface 130, the bright area below it represents the plasma 136 through which light is transmitted, and the dark area at the bottom is where the light is blocked by the red blood cells 140.

FIG8B shows that to extract the locations of the air/plasma interface and the plasma/red blood cell interface, the first derivative of the interface with respect to time can be determined (edge detection). The derivative is relative to the distance down the tube (y-axis) rather than time (x-axis). FIG8B shows the locations of the air/plasma (upper) interface 130 and the plasma/red blood cell (lower) interface 132.

In one non-limiting example, the positions of two local maxima of the image in Figure 8B are determined, one local maximum representing the air/plasma interface and the other representing the plasma/red blood cell interface. To convert these (pixel) positions into the volume occupied by the entire sample and the volume occupied by the red blood cells, the y positions of the top and bottom of the centrifuge tube (such as recorded from the still tube image shown in Figure 2) are used as reference positions along with knowledge of the shape of the centrifuge vessel.

9 , the plasma/erythrocyte interface position is converted to the volume fraction occupied by red blood cells and plotted against time as a centrifuge-assisted sedimentation curve 180. This curve in FIG9 is the result of one non-limiting example of a centrifuge-based method of determining a sedimentation curve extracted from a video recording.

Calculation of ESR from sedimentation curves

Once the sedimentation curves of Figure 9 are obtained for each sample, there are many possible ways to extract parameters related to the ESR. A simple way to reduce the curve to a single parameter for analysis is to fit a single exponential to the curve at the plasma/erythrocyte interface using a standard nonlinear least squares fit.

One such example is shown in FIG10A . For FIG10A , the data in the graph is shown as black dots 200 , the x-axis is time in seconds, and the y-axis is the volume fraction occupied by red blood cells. A single exponential fit is shown as line 202 .

Referring now to Figure 10B, the data in this graph is shown as black dots 200. Figure 10B shows a substantially bilinear fit. The gradient of the initial linear portion shown by the linear fit 210 can be determined, as well as the time 212 of the transition between the initial linear segment and the nonlinear region where packing slows, shown here by the red line 214.

While these simple techniques using standard nonlinear least squares fitting can yield information about the ESR, when such measurements are compared to traditional Westergren ESR measurements, correlations based on nonlinear least squares (NLS) fitting leave room for improvement because NLS itself does not take into account certain correction coefficients.

Effects of plasma proteins on ESR

Understanding some of the factors that may affect ESR measurements helps to extract ESR parameters that correlate more closely with traditional Westergren ESR measurements. The parameter of interest (ESR) responds to the concentration of certain plasma proteins and can be directly influenced/manipulated by adding one of these proteins to the blood sample.

In this example, as a technique to provide samples with a wide range of ESR values, exogenous fibrinogen was used to create blood samples with ESR values spanning the entire range of interest (0-120 mm/h in the Westergren method). Figure 11 shows how the addition of fibrinogen increases the Westergren ESR value.

As shown in Figures 11 to 14, several parameters from the centrifugation analysis show good correlation with fibrinogen levels (and therefore good correlation with ESR), the most significant time constant according to the single exponential fit, the time of onset of packing, and the initial linear gradient. With reference to Figures 12, 13, and 14, in some embodiments, each of these parameters can be used to obtain an estimate of the Westergren ESR value. Both the single exponential fit time constant and the packing start time have the advantage of being independent of the y-axis scale. The packing start time and the initial linear gradient have the advantage of having clear physical meaning.

Figure 11 shows that Westergren ESR values increase with increasing added fibrinogen. Figure 11 illustrates a single sample with different levels of fibrinogen added thereto.

Figure 12 shows the time constant of the monoexponential fit of the original sedimentation curve, which showed a good correlation with the level of added fibrinogen.

FIG13 shows the time of onset of cell packing, which showed a good correlation with the level of added fibrinogen.

Figure 14 shows the initial linear gradient of the raw sedimentation curve, which showed a good correlation with the level of added fibrinogen.

Effect of hematocrit on ESR

It should be understood that, in addition to fibrinogen, hematocrit is another factor that affects Westergren and other ESR measurements. In fact, Westergren erythrocyte sedimentation is strongly affected by hematocrit. In the Westergren method, many laboratories either do not report results for samples with a hematocrit greater than about 45%, or adjust the sample hematocrit to a fixed level (usually 45%) before measuring the ESR. This embodiment of the method is actually superior to the Westergren technique in that the Westergren is saturated (i.e., does not react to fibrinogen <10 mg/ml), while this embodiment of the method is not saturated except for 15 mg/ml.

Centrifuge-based ESR sedimentation is even more strongly affected by hematocrit levels than measurements under gravity. For at least some embodiments herein, the increased reliance on hematocrit is due to lower volume—and therefore smaller vessel size. Increasing hematocrit, which typically means red blood cells start closer together, increases blood viscosity by presenting a physical barrier to free movement, and reduces the maximum distance an interface can travel before cells become packed, all of which lowers the ESR, independent of fibrinogen from inflammation.

To illustrate the significant confounding effect of hematocrit, centrifuge-based ESR measurements, performed by taking the same blood sample and adjusting the hematocrit before measuring the ESR, showed that a person with a typical hematocrit of 45% and a normal ESR of 22 mm/h would exhibit an ESR of 5 mm/h (very low) if the hematocrit was 60%, but would exhibit an ESR of 93 mm/h (very high) if the hematocrit was 35%, even in the absence of changes in the levels of clinically important plasma proteins. In other words, the change in ESR due to hematocrit may be greater than the change in ESR due to plasma proteins of interest to clinicians.

There are several traditional approaches to compensating for this confounding effect of hematocrit. One approach is to use a hematocrit compensation curve, such as that from Dintenfass (1974). A more accurate (if more labor-intensive) way to eliminate the confounding effect is to simply change the hematocrit to a standard value before testing, rather than using a chart to correct for the hematocrit. Some ESR techniques, such as "hematocrit-corrected ESR," include an initial step that fixes the hematocrit to a set value (e.g., 45%) so that the measured ESR truly reflects the protein content of the plasma (clinically relevant), rather than the hematocrit (Borawski and 2001).

As seen in Figure 15, to understand and estimate the effect of hematocrit, a set of eleven (11) samples were adjusted to 35%, 45%, and 55% hematocrit and then examined by centrifuge ESR and Westergren ESR techniques. The correlation of the centrifuge monoexponential time constant and the Westergren ESR is shown for the hematocrit-adjusted clinical blood samples. The samples correlated well within each hematocrit, but did not correlate well across all hematocrit samples.

Referring now to FIG. 16 , the position of the plasma/erythrocyte interface is plotted as a function of time for different clinical samples with varying hematocrit levels for a centrifuge-based ESR experiment. Several blood samples with unadjusted and adjusted fibrinogen levels and hematocrit are shown: red squares: 35%, green triangles: 45%, and blue diamonds: 55% hematocrit. FIG. 16 shows the complete sedimentation profile, while FIG. 17 shows the sedimentation profile for a sample adjusted to a given hematocrit over a shorter period (<10 seconds) during the initial measurement. The sample's sedimentation profile shows a sharp drop during the initial measurement period, accompanied by an almost linear decrease in the position of the interface over time during the initial measurement period. Sedimentation rates then slow as the red blood cells pack together. A number of data sets corresponding to various hematocrits (as indicated) and various ESR rates are shown in FIG. 16 .

In Figure 17, which shows sedimentation over a short time period (<10s), the high quality of the data obtained with such a short measurement time shows a linear sedimentation rate for all hematocrit levels during the initial period. In one embodiment described herein, the linear region of the sedimentation profile can be used to extract the sedimentation velocity. The raw sedimentation velocity is plotted against the Westergren ESR. Assuming that the three fitted lines corresponding to the three hematocrit levels in Figure 17 are discontinuous, it is desirable to compensate for the hematocrit to derive a clinically effective ESR value from the raw value.

As a further example, FIG18A shows the logarithm of the red blood cell sedimentation rate extracted from the sedimentation profile (uncorrected for hematocrit). FIG18A also shows that the centrifuge-based sedimentation rate is strongly dependent on the hematocrit, even more so than the Westergren-based sedimentation rate. The narrow cross-section of the centrifuge tube increases the hydrodynamic resistance to fluid flow due to the red blood cells. The centrifugation process involves the flow of plasma through a bed of red blood cells, which provides hydrodynamic resistance. This resistance is a function of the volume fraction of red blood cells (hematocrit).

In order to obtain a better correlation between the centrifuge-based sedimentation rate and the Westergren sedimentation rate, the centrifuge-based sedimentation rate was corrected for the effect of hematocrit. The correction used can be expressed as follows,

Where U _uncorr and U _corr are the uncorrected (raw) and corrected sedimentation rates, respectively, is the volume fraction of cells (hematocrit), and γ is an empirical parameter obtained by curve fitting, such as described below or developed in the future. The correction factor represents a simple mathematical form to account for the increased drag force exerted by red blood cells. It should be understood that while this functional form has been found to correct for hematocrit, other functions will also work.

For non-limiting example, one way to calculate γ is by a calibration technique, such as, but not limited to, the following technique: for a diverse set of samples (different hematocrits, ESR values, etc.), the ESR values are determined using a reference method and by a centrifuge-based method. The γ and γ parameters are determined as a calibration for each centrifuge setting and can be changed at least in part based on the vessel geometry and sample volume. Therefore, if at least one of these factors is changed, the parameters may need to be recalculated. For a centrifuge setting as described herein, the optimal values of these parameters were found to be: γ and γ = 3.85. It should be understood that these parameters are used for fit optimization and are not directly related to physical parameters. It should be understood that this is merely a non-limiting example and that other curve fitting or similar techniques can be used.

Hematocrit Measurement Techniques

For purposes of calculating the hematocrit correction factor, it will be appreciated that the hematocrit value may be known prior to the centrifuge-based sedimentation test, and in such cases, a corrected ESR result can be quickly obtained based on the initial linear portion of sedimentation and the known hematocrit level, without having to wait until the red blood cells have been fully compacted by centrifugation. Alternatively, some embodiments may determine the hematocrit level during or after centrifugation.

Hematocrit measurements performed before, during, or after centrifugation by non-centrifugation methods include at least the following. One technique involves measuring hemoglobin concentration. For example, in approximately 99% of the population, there is a 1:1 correlation between hemoglobin measurements and hematocrit levels. Therefore, if hemoglobin test data is available, the hematocrit level is typically known before centrifuge-based sedimentation testing.

Referring now to FIG18C , one embodiment of an assay protocol for hemoglobin-based hematocrit measurement will now be described. Blood is diluted 1:100 with water. The diluted sample is mixed (1:3) with modified Drabkin reagent (Sigma D5941, containing sodium bicarbonate, potassium ferricyanide, and potassium cyanide supplemented with 0.015% polyoxyethylene fatty alcohol ether (Brij) 35). After 10 minutes at 37° C., the absorbance of the reaction product (cyanomethemoglobin) is measured at 540 nm. The assay is calibrated with bovine hemoglobin (Sigma 2500), which produces a linear dose response over the range of 0-20 g/dL.

The correlation of the results with hematocrit measurements will now be discussed using the assay protocol for hemoglobin-based measurements. Human blood samples were processed by recombining plasma and red blood cells (collected by centrifugation) to provide a wide range of hematocrit values. These samples were assayed as described above and by standard centrifugation capillary hematocrit assay, and the results are shown below. As shown in Figure 18C, the resulting correlation is accurate, with slope = 1, intercept = 0, and correlation coefficient (R^2) = 0.99.

Another technique for hematocrit measurement involves microscopic imaging. Hematocrit can also be measured in an apparatus using a cuvette of fixed depth and a blood sample diluted to a known degree. A description of a system having such a cuvette is found in U.S. patent application Ser. No. 13/244,947, which is incorporated herein by reference in its entirety for all purposes. Hematocrit can be determined by microscopic measurement of: (1) red blood cell count per field of view and (2) mean corpuscular volume. The preferred methods are: (1) dark field microscopy and (2) (1) + fluorescence microscopy using fluorescently labeled anti-human CD-35 (red blood cell surface antigen). Image analysis techniques are then applied.

Specifically, a method for measuring hematocrit may involve measuring the optical density of a sample. See, for example, Lipowsky et al., "Hematocrit determination in small bore tubes from optical density measurements under white light illumination," Microvascular Research, Vol. 20, No. 1, July 1980, pp. 51–70 ; http://dx.doi.org/10.1016/0026-2862(80)90019-9 , which is incorporated herein by reference in its entirety for all purposes. Lipowsky discusses the relationship between the hematocrit of blood flowing in small bore glass tubes that has been measured for various lumen diameters and its optical density (OD) under white light (tungsten lamp) illumination. In at least some embodiments herein, all of this data is available because a small bore tube of blood is being illuminated.

In another embodiment, the hematocrit level can be determined by examining a portion of the blood sample under microscopic or other magnified observation, such as, but not limited to, measuring the number and average size of red blood cells within a defined observation area, which may have a known size. In this manner, the hematocrit level can be determined based on such visual characteristics of the red blood cells.

In yet another example, the hematocrit level can be determined based on a complete centrifugation of a blood sample that has been compacted with red blood cells. This level of compaction can be used to determine the hematocrit. In this example, only the linear portion of the centrifuge-based sedimentation test is used to determine the corrected ESR. By way of non-limiting example, a first, linear, initial portion of the interface position measurement, along with a final, also linear, end portion, are two portions of the sedimentation that can be used to calculate the ESR corrected for hematocrit. As seen in FIG16 , this non-limiting example can use a linear portion 182 of the sedimentation curve corresponding to the initial period after centrifugation and another linear portion 184 of the sedimentation curve near the end (when compaction is essentially complete and the curve is substantially flat). The non-linear portion of the sedimentation curve between the linear portions 182 and 184 is essentially not used to calculate the hematocrit correction factor.

The foregoing is a non-exhaustive list of hematocrit calculation techniques and does not exclude other methods for measuring hematocrit levels for use with the sedimentation measurement techniques described herein.

In a non-limiting example of how this works together to perform ESR measurements, a vessel containing a sample can be centrifuged under controlled conditions for a first time period, such as, but not limited to, a time period associated with the linear portion of the sedimentation curve. In a non-limiting example, centrifugation is controlled to a specific force range (such as the rpm range of a centrifuge's rotor) such that the force applied to the accelerated sedimentation process is substantially consistent. The sedimentation profile during the initial time period, as previously described, is typically linear, and it may be desirable to capture a sedimentation image of the formed components in the material after centrifugation during the initial linear time period. For non-limiting examples, images can be taken in several scenarios, such as, but not limited to: a) when the vessel is in a centrifuge, b) when the vessel is in a centrifuge but the centrifuge stops, or c) by removing the vessel from the centrifuge and imaging it. In some embodiments, sedimentation can be measured with a single image. It should be understood that the level at start time t0 is the meniscus level of the remaining solution above the supernatant or precipitated formed components. The level of sedimentation is the level of precipitated forming components in the image after an initial period of accelerated sedimentation.

The hematocrit measurement for ESR calculation can be performed using at least one method such as, but not limited to, those methods described herein. It can be performed in the same system as a system with a centrifuge, or alternatively, it can be performed using a physically separate instrument. In a non-limiting example, after obtaining an image of the sedimentation, the sample can be centrifuged to complete the sedimentation and the formed components can be compacted into granular matter. The effective gravity on the vessel can completely cause the precipitate ("granular matter") to accumulate on the bottom of the tube. The supernatant liquid is then poured out from the tube without disturbing the precipitate, or the supernatant liquid is aspirated with a pipette.

Graph of ESR corrected for hematocrit

Figure 18B shows the logarithm of the erythrocyte sedimentation rate extracted from the sedimentation curve corrected for the effect of hematocrit. As can be seen from the improvement in the correlation coefficient, hematocrit correction (e.g., see Figure 19A) can substantially eliminate the effect of hematocrit on ESR.

Using clinical samples adjusted for hematocrit, a good correlation with ESR was found within each hematocrit level, and, as expected, a significant effect of hematocrit was also found. Centrifugation can also be used to obtain accurate values for hematocrit and to correct for the effect of hematocrit.

FIG19 shows a replot of the data of FIG18B in which the effect of hematocrit is clearly minimized as illustrated by the good correlation of the values of hematocrit-corrected ESR (present method) and ESR according to the traditional Westergren test technique.

Referring now to Figure 20, however, the hematocrit-corrected ESR of this method does not have a linear relationship with the Westergren ESR, as can be seen in Figure 19. To obtain an estimate of sedimentation velocity that is linearly related to the Westergren ESR, the hematocrit-corrected data from the centrifuge can be further corrected using the following formula:

Estimated Westergren ESR = 10Λ(((LOG(HCT corrected ESR) - LOG(644.11))/0.1367)), where the relationship and parameters used are those derived from analysis of FIG. 18B .

FIG20 shows hematocrit-corrected and linearly transformed Log(ESR) values obtained using this embodiment, compared to Westergren ESR (uncorrected hematocrit). In FIG20 , a calibration (based on the fit calculated as per FIG19 ) has been applied, and the agreement between Westergren and this method is shown. The reference line in this plot is the y=x line. FIG20 shows an example of accuracy.

Experimental methods

The data obtained for the various graphs were obtained using the following techniques. These are provided as examples and are not intended to be non-limiting.

Samples : Fresh EDTA-anticoagulated blood samples were used. EDTA was used because it is the standard for the "modified Westergren" method. Samples were kept at room temperature and resuspended before measurement.

Hematocrit adjustment : The sample is spun down to allow for hematocrit (e.g., 5000 relative centrifugal force (RCF) for 20 minutes) and to separate the plasma from the cells. The red blood cells are slurried with plasma from the same sample and more plasma is added to obtain the desired hematocrit level.

Westergren ESR measurement: 1 mL of sample is required for Westergren ESR measurement (using 'Sedigren' brand tubes, following the protocol enclosed therein). Red blood cell sedimentation is observed and measured by video recording.

Modulation of RBC Zeta Potential (and ESR) with Fibrinogen: For the examples shown in Figures 11-14, bovine fibrinogen was dissolved in blood. In one example, for a 40% hematocrit sample, a range of 0-10 mg/mL produced an ESR range of 5-100 mm/h.

Measurement of Centrifuge Sedimentation Curve: A 25 uL sample of whole blood is added to a centrifuge vessel. A swing bucket centrifuge as described in co-pending U.S. patent application serial numbers 13/355,458 and 13/244,947 is modified to have slots cut to allow light to pass through the bucket while the bucket is rotating horizontally (axis of rotation vertical). In this non-limiting example, the light source can be a 1 W green LED, such as available from Thorlabs of Newton, New Jersey, with the brightness adjusted (typically ~10%) so that the light reaching the detector does not saturate it. A webcam or other imaging device (such as available from Logitech) is positioned 10 mm above the plane of rotation as shown in FIG3 . The integration time is 200 ms. Images are captured at 5 frames per second (fps) for a known period of up to three minutes using a lossless compression codec ("huffyuv").

Image Transformation : Images obtained from visual observation of the centrifuge vessel during centrifugation were processed in the manner described herein for Figures 6A-7C.

Sedimentation Curve Extraction : The position of the red blood cell/plasma interface and other interfaces in the image are then plotted over time in the manner described herein for Figures 8A-9.

Curve Fitting with Hematocrit Correction Factors : The sedimentation curves were then further processed to obtain sedimentation rate information by curve fitting using the various techniques described herein for Figures 10A-10B, with or without the use of hematocrit correction factor(s).

Measurement of non-red blood cell blood components

While this specification is primarily written in the context of measuring erythrocyte sedimentation rate, it should be understood that the techniques herein can be adapted for use in measuring sedimentation rates of other formed blood components other than erythrocytes. Some embodiments can measure platelet sedimentation. Some embodiments can measure white blood cell sedimentation. Alternatively, sedimentation of other formed components can also be measured.

21 , a kymogram obtained using a centrifuge-based method as described herein also shows the presence of a "shadow" showing a white blood cell and plasma interface 141, in addition to the air/plasma interface 130 and the red blood cell/plasma interface 132. Thus, both the red blood cells preceding the red blood cells and a preceding secondary sedimentation corresponding to the white blood cells are observed in the sedimentation kymogram of FIG21 .

Therefore, as shown in Figure 21, some embodiments of centrifugation can be used to measure the white blood cell sedimentation rate (WCS) sequentially or simultaneously, which may be useful in characterizing certain aspects of patient health. For example, when white blood cells are activated and/or aggregate, they change their physical properties. Both phenomena are of great interest in assessing white blood cell function. White blood cells settle under centrifugal force, but they settle at a slower rate than red blood cells. The rate at which white blood cells settle is a function of at least one of the following: WCS density, shape, and aggregation state. Measuring sedimentation rate can result in detection of one or more of these changes, which in turn can be used to characterize certain aspects of patient health.

By way of non-limiting example, it will be appreciated that changes in refractive index or possibly light scattering can be used as a measure of the location of blood component interfaces, rather than changes in absorbance. Alternatively, some embodiments may use both. FIG. 21 shows data indicating that a white blood cell interface is detectable due to changes in refractive index or light scattering, rather than changes in absorbance. In one embodiment, the location of the RBC interface is based on changes in absorbance due to hemoglobin's significant absorption in the green portion of the wavelength spectrum. If light of the correct wavelength (very long wavelength) is used, it may be possible to similarly monitor the RBC interface. Thus, the use of light scattering or changes in refractive index can also be used, alone or in combination with absorbance, as an alternative to measuring interface location or to detect certain interface(s), such as white blood cells or platelets, that are not readily visible through absorbance detection alone.

Assay processing in integrated automation systems

With reference now to Figure 22, it should be understood that the process described herein can be performed using automation technology. Automated processing can be used in an integrated automated system. In some embodiments, this can be in a single instrument having multiple functional components and surrounded by a common housing. The processing techniques and methods for sedimentation measurement can be preset. Alternatively, it can be based on a scheme or program that can be dynamically changed as needed in the manner described in U.S. Patent Application Serial Nos. 13/355,458 and 13/244,947, and the above-mentioned documents are all incorporated herein by reference in their entirety for all purposes.

In one non-limiting example, as shown in FIG22 , an integrated instrument 500 may be equipped with a programmable processor 502 that can be used to control various components of the instrument. For example, in one embodiment, the processor 502 can control a single or multiple pipette systems 504 that can move in the X-Y and Z directions, as indicated by arrows 506 and 508. The same processor or a different processor can also control other components 512, 514, or 516 in the instrument. In one embodiment, the components 512, 514, or 516 include a centrifuge.

As shown in Figure 22, controlling by processor 502 can allow the sample handling system such as but not limited to pipette system 504 to collect samples from cartridge 510 and move the sample to one of assembly 512, 514 or 516. In a non-limiting example, sample is blood. Such movement can relate to sample distribution in the detachable vessel in cartridge 510 and then transport the detachable vessel to one of assembly 512, 514 or 516. Alternatively, sample is directly distributed in a container installed in one of assembly 512, 514 or 516. Alternatively, this can occur without sample distribution in a container at one of assembly. Alternatively, some embodiments can use a container for carrying out sample collection from the experimenter and process the sample when sample is still in the collection vessel. This allows the sample collected in the container to be directly transported to assembly, such as but not limited to centrifuge or other sample separators, without further sample fluid being transferred to another container used in centrifuge device. Alternatively, the container in which the sample is collected from the subject can have a shape that is conducive to enabling a small volume blood sample to be centrifuged. Alternatively, this collection container can be sufficiently transparent on a portion of at least one surface to allow imaging of the sample therein without the need to remove the sample from the collection container. Alternatively, this collection container can be sufficiently transparent on the alignment portion of at least two surfaces of the container to allow imaging of the sample therein without the need to remove the sample from the collection container. In such a non-limiting example in which the ESR method is performed on the collection container, there is no sample loss associated with a portion of the sample being divided equally into separate containers for ESR measurement. In this non-limiting example, the method for processing the sample may involve removing the collection vessel from the sample collection device, optionally the collection vessel is transported to the sample processing unit, and optionally the collection vessel is inserted into a cartridge or directly inserted into one of the components. Alternatively, some methods can transport the collection vessel to a pre-treatment unit for pre-treatment, then the collection vessel or the cartridge with the collection vessel is loaded into the sample processing unit. In one non-limiting example, a method for processing a sample can involve removing a collection vessel from a sample collection device, followed by transporting the collection vessel to a sample processing unit. In an embodiment, a method for processing a sample can involve removing a collection vessel from a sample collection device, followed by transporting the collection vessel to a sample processing unit, followed by inserting the collection vessel into a cartridge or directly into one of the components. For non-limiting example, the sample processing unit can be, but is not limited to, those described in U.S. Patent Applications 13/769,820 and 61/852,489, both of which are incorporated herein by reference in their entirety for all purposes.

In a non-limiting example, one of these assemblies 512, 514 or 516 can be a centrifuge with an imaging configuration as shown in Figure 3. Other assemblies 512, 514 or 516 perform other analyses, determinations or detection functions. In a non-limiting example, the sample vessel in the centrifuge of one of these assemblies 512, 514 or 516 can be moved to another (or alternatively another position or device) in assembly 512, 514 or 516 by one or more manipulators from one of assembly 512, 514 or 516, so that sample and/or sample vessel are further processed. Some embodiments can use pipette system 504 to engage sample vessel to move it from assembly 512, 514 or 516 to another position in the system. In a non-limiting example, this is for moving the sample vessel to an analysis station (such as but not limited to imaging), then the vessel is moved back to the centrifuge so that further processing may be useful. In an embodiment, this can be completed using pipette system 504 or other sample handling systems in the device. In one non-limiting example, movement of vessels, tips, etc. from cartridge 510 to one of components 512, 514, or 516 to another location in the system (or vice versa) can also be accomplished using pipetting system 504 or other sample handling system in the device.

All of the foregoing can be integrated into a single housing 520 and configured for countertop mounting or small footprint floor mounting. In one example, a small footprint floor mounted system can occupy a floor area of approximately 4 m ² or less. In one example, a small footprint floor mounted system can occupy a floor area of approximately 3 m ² or less. In one example, a small footprint floor mounted system can occupy a floor area of approximately 2 m ² or less. In one example, a small footprint floor mounted system can occupy a floor area of approximately 1 m ² or less. In some embodiments, the instrument footprint can be less than or equal to about 4 m ² , 3 m ² , 2.5 m ² , 2 m ² , 1.5 m ² , 1 m ² , 0.75 m ² , 0.5 m ² , 0.3 m ^{2 ,} 0.2 m ² , 0.1 m ² , 0.08 m ² , 0.05 m ² , 0.03 m ² , 100 cm 2 , 80 cm ² , 70 cm ² , 60 cm ² , 50 cm ² , 40 cm ² , 30 cm ² , 20 cm ² , 15 cm ² , or 10 cm ² . Some suitable systems in a point of service environment are described in U.S. patent application serial numbers 13/355,458 and 13/244,947 , all of which are incorporated herein by reference in their entirety for all purposes. The present embodiment may be configured for use with any of the modules or systems described in these patent applications.

Although the present invention has been described and illustrated with reference to certain specific embodiments thereof, it will be understood by those skilled in the art that various modifications, changes, amendments, substitutions, deletions or additions to the procedures and protocols may be made without departing from the spirit and scope of the present invention. For example, with any of the above-described embodiments, it will be understood that other techniques for plasma separation may also be used in conjunction with or in place of centrifugation. For example, one embodiment may centrifuge the sample for an initial period of time, after which the sample may be positioned in a filter that then removes the formed blood components to complete the separation. Although the present embodiment is described in the context of centrifugation, other accelerated separation techniques may also be suitable for use with the sedimentation rate measurement methods described herein. Some embodiments may optionally combine the hematocrit correction techniques described herein with measurement techniques such as those described in U.S. Patent No. 6,204,066, which is incorporated herein by reference in its entirety for all purposes. Some embodiments herein may pre-treat the blood sample to pre-set the hematocrit value in the sample to a predetermined value in order to remove the variable due to hematocrit. Some embodiments may also use conventional techniques for adjusting the hematocrit level. It should be understood that while the present embodiments are described in the context of blood samples, the techniques herein may also be configured for application to other samples (biological or otherwise).

Alternatively, at least one embodiment can use a variable speed centrifuge. Utilize feedback, such as but not limited to the imaging of the position of (one or more) interfaces in the sample, the speed of the centrifuge can be changed to keep compaction curve and time (until complete compaction), and the ESR data extracted from the velocity profile of the centrifuge rather than the sedimentation rate curve are linear. In such a system, one or more processors can be used to feedback control the centrifuge to have a linear compaction curve while also recording the velocity profile of the centrifuge. According to which interface is being tracked, sedimentation rate data are calculated based on centrifuge speed. In a non-limiting example, when compaction is near completion, a higher centrifuge speed is used to keep a linear curve.

In addition, those skilled in the art will recognize that any embodiment of the present invention can be applicable to the collection of sample fluid from human, animal or other experimenter.Alternatively, the volume of blood for sedimentation test can be 1mL or less, 500 μ L or less, 300 μ L or less, 250 μ L or less, 200 μ L or less, 170 μ L or less, 150 μ L or less, 125 μ L or less, 100 μ L or less, 75 μ L or less, 50 μ L or less, 25 μ L or less, 20 μ L or less, 15 μ L or less, 10 μ L or less, 5 μ L or less, 3 μ L or less, 1 μ L or less, 500nL or less, 250nL or less, 100nL or less, 50nL or less, 20nL or less, 10nL or less, 5nL or less or 1nL or less.In some embodiments, the sample collected herein comprises capillary blood. In some embodiments, the sample is collected from a finger prick. In some embodiments, the sample is collected from the skin at an alternative site such as a forearm, leg, earlobe or other positions on the subject. Alternatively, some embodiments can use a collection process, wherein the target area is warmed and/or wherein the initial blood sample is wiped off (or not wiped off) (and at least a portion of the remainder is collected for processing). Capillary blood can be collected by capillary, with a syringe or by the capillary mode of a collection container. Alternatively, in some embodiments, the sample collected herein mainly comprises capillary blood with negligible tissue fluid. Alternatively, some embodiments can use an integrated collection device with multiple collection tubes or chambers, wherein only a subset of the tubes or chambers is imaged for ESR. Alternatively, some embodiments can use an integrated collection device with multiple collection tubes or chambers, wherein only one of the tubes or chambers is imaged for ESR.

In addition, concentration, amount and other numerical data can be presented in range format in this article.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value that is clearly stated as range limit, but also including the single numerical value or sub-range contained in the range, as if each numerical value and sub-range are clearly stated.For example, the size range of about 1nm to about 200nm should be interpreted as not only including the limit of about 1nm and about 200nm that is clearly stated, but also including a single size, such as 2nm, 3nm, 4nm, and sub-range, such as 10nm to 50nm, 20nm to 100nm etc.

The publications discussed or referenced herein are provided only for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Furthermore, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the structures and/or methods associated with the referenced publications. The following applications are incorporated herein by reference in their entirety for all purposes: U.S. Patent Application Serial Nos. 13/355,458, 13/244,947, 13/769,820, 61/852,489, U.S. Provisional Application Serial No. 61/673,037, filed on July 18, 2012, entitled “Rapid Measurement of Formed Blood Component Sedimentation Rate from Small Sample Volumes”; U.S. Provisional Application Serial No. 61/673,037, filed on January 22, 2014 No. 61/930,432, filed on July 26, 2012; U.S. Patents 8,380,541, 8,088,593; U.S. Patent Publication No. 2012/0309636; U.S. Patent Application Serial No. 61/676,178, filed on July 26, 2012; PCT/US2012/57155, filed on September 25, 2012; U.S. Application Serial No. 13/244,946, filed on September 26, 2011; U.S. Patent Application 13/244,949, filed on September 26, 2011; and U.S. Application Serial No. 61/673,245, filed on September 26, 2011.

The following paragraphs list various aspects of at least some of the embodiments described herein:

Aspect 1. A method comprising: applying an accelerated blood component separation technique to a blood sample for a period of time to separate formed blood components from plasma; establishing a time-dependent compaction curve for at least one formed blood component in the blood sample after the accelerated blood component separation has begun, the compaction curve having an initial approximately linear portion; and determining a sedimentation rate of the formed blood component based at least on: the compaction curve and a hematocrit correction factor.

Aspect 2. A method comprising: centrifuging a blood sample in a vessel for a period of time; establishing a time-dependent compaction curve for at least one formed blood component in the blood sample after centrifugation has begun, the compaction curve having an initial approximately linear portion; and correcting the effect of hematocrit on the sedimentation rate of the formed blood component by applying a hematocrit correction factor to the approximately linear portion of the compaction curve.

Aspect 3. A method comprising: centrifuging a blood sample in a vessel for a period of time; establishing a time-dependent compaction curve for at least one formed blood component in the blood sample after centrifugation has begun; correcting for the effect of hematocrit on the sedimentation rate of the formed blood component using a hematocrit correction factor based on the formula:

where U _uncorr and U _corr are the uncorrected (raw) and corrected sedimentation rates, is the volume fraction of cells (hematocrit), and and γ are empirical parameters obtained by curve fitting.

Aspect 4. The method of any of the above aspects, wherein the curve fitting for the hematocrit correction factor comprises calibrating the sedimentation rate according to the centrifuge-based technique with the sedimentation rate according to the reference technique.

Aspect 5. The method according to any one of the above aspects, wherein the reference technique is the Westergren technique.

Aspect 6. The method of any one of the above aspects, wherein fibrinogen levels up to 15 mg/ml do not affect sedimentation rate measurements.

Aspect 7. The method of any one of the above aspects, wherein the blood sample is about 100 uL or less.

Aspect 8. The method of any one of the above aspects, wherein the blood sample is about 50 uL or less.

Aspect 9. The method of any one of the above aspects, wherein the blood sample is about 25 uL or less.

Aspect 10. The method of any of the above aspects, wherein centrifugation occurs at a first speed for a first period of time and then at a second, faster speed for a second period of time.

Aspect 11. The method of any of the above aspects, wherein the centrifugation uses a centrifuge configured to allow visual observation of the blood sample during centrifugation to establish the interface location of one or more formed blood components in the blood sample.

Aspect 12. The method of any one of the above aspects, wherein the centrifugation uses a centrifuge having a window thereon to enable visual observation of the blood sample to establish the position of the red blood cell/plasma interface over time.

Aspect 13. A method as described in any of the above aspects, wherein the centrifugation uses a centrifuge, a light source and an image capture device to enable visual observation of the blood sample to establish the position of the formed blood component/plasma interface over time.

Aspect 14. The method of any one of the above aspects, wherein the compaction curve data is collected by capturing a plurality of images of interface locations of one or more formed blood components in the centrifuge vessel during the time period.

Clause 15. The method of clause 14, wherein pixel positions in the plurality of images are used to accurately determine the interface position.

Aspect 16. The method of aspect 14, wherein capturing of images begins once the centrifuge has reached a minimum operating speed.

Aspect 17. The method of aspect 14, wherein the capturing of the image begins when the centrifuge has begun to rotate.

Aspect 18. The method of any one of the preceding aspects, wherein the compaction curve data is collected while the sample is being centrifuged.

Aspect 19. The method of any of the above aspects, wherein centrifugation is used to obtain an accurate value of the hematocrit and to correct for the effect of hematocrit on sedimentation rate measurements.

Aspect 20. A method as described in any of the above aspects, wherein the correction for hematocrit includes calculating a mathematical function for the locations of multiple formed blood component interfaces appearing in the curve, which function can be used to correct for changes in sedimentation rate due to hematocrit.

Aspect 21. The method of any of the above aspects, wherein the hematocrit correction factor is determined without using data from a non-linear portion of the compaction curve.

Aspect 22. The method of any of the above aspects, wherein the hematocrit level in the sample is obtained from a technique separate from centrifugation.

Aspect 23. The method according to any one of the above aspects, wherein and γ are used for fitting optimization without being directly related to physical parameters.

Aspect 24. The method according to any one of the above aspects, further comprising image transformation for converting a curved interface to a flat interface.

Aspect 25. The method of any of the above aspects, wherein the hematocrit correction is capable of substantially eliminating the effect of hematocrit on the sedimentation rate of formed blood components.

Aspect 26. A method as described in any of the above aspects, wherein image transformation parameters are selected so that a video of the position of the interface of the formed blood components is subjected to image transformation, and then a region of interest covering the entire position range of both the air/plasma interface and the red blood cell interface is selected.

Aspect 27. A method as described in any of the above aspects, wherein for each time point in the video, the pixel intensity values across each row of the sample vessel within the region of interest are averaged to produce a single column representing the intensity radially down the sample vessel.

Aspect 28. The method of any one of the above aspects, wherein the columns for each time point are then assembled into a kymogram.

Aspect 29. The method of aspect 28, wherein the positions of two local maxima of the image are determined, one local maximum representing the air/plasma interface and the other representing the plasma/red blood cell interface.

Aspect 30. The method of Aspect 28, comprising converting pixel positions into the volume occupied by the entire sample and the volume occupied by red blood cells, wherein the y positions of the top and bottom of the centrifuge vessel are used as reference positions together with knowledge of the shape of the centrifuge vessel.

Aspect 31. The method of any one of the preceding aspects, comprising converting the plasma/erythrocyte interface position into said volume fraction occupied by red blood cells and plotting it as a centrifuge sedimentation curve versus time.

Aspect 32. The method of any one of the above aspects, wherein the linear region of the sedimentation profile is used to extract the sedimentation rate.

Aspect 33. A method as described in any of the above aspects also includes obtaining an estimate of the sedimentation rate that is linearly related to the Westergren ESR, and the data obtained from the centrifuge and corrected for hematocrit are further corrected using the following formula: estimated Westergren ESR = 10^(((LOG(ESR corrected for HCT)-LOG(644.11))/0.1367)).

Aspect 34. The method of any of the above aspects, further comprising performing hematocrit correction and linear transformation on the Log(ESR) values to create a linear plot of sedimentation velocity.

Aspect 35. The method of any one of the above aspects, wherein the blood sample is whole blood.

Aspect 36. The method of any one of the above aspects, wherein the blood sample is an anticoagulated sample.

Aspect 37. The method of any one of the preceding aspects, wherein the formed blood components are white blood cells.

Aspect 38. The method of any of the above aspects, wherein the formed blood components are platelets.

Aspect 39. The method of any of the above aspects, further comprising determining a leukocyte sedimentation rate after centrifugation has begun, wherein measuring the leukocyte sedimentation rate characterizes at least one of the following regarding the white blood cells: cell density, shape, and aggregation state.

Aspect 40. A method comprising: collecting multiple images of the interface locations of formed blood components and plasma over time from an accelerated blood sample compaction process; performing an image transformation on the multiple images to transform images having curved interfaces into corrected images having straight linear interfaces; and establishing a time-dependent compaction curve for at least one formed blood component in the blood sample based on the interface locations in the corrected images.

Aspect 41. A method comprising: centrifuging a blood sample in a vessel for a period of time; collecting multiple images of the interface position of formed blood components and plasma over time; performing an image transformation on the images to transform an image having a curved interface into a corrected image having a straight linear interface; and establishing a time-dependent compaction curve for at least one formed blood component in the blood sample based on the interface position in the corrected image after centrifugation has begun.

Aspect 42. A method comprising: transferring at least a portion of a blood sample from a blood sample location to a centrifuge vessel using a programmable processor-controlled system; transferring the vessel from a first addressable location to a centrifuge having a second addressable location using a sample handling system controlled by the programmable processor; centrifuging the blood sample in the vessel for a period of time; collecting a plurality of images of a location of an interface between formed blood components and plasma over time;

A time-dependent compaction curve is established for at least one shaped blood component in the blood sample based on the position of the interface in the corrected image after centrifugation has begun.

Aspect 43. The method of any of the above aspects, wherein the centrifuge has a rotor with a diameter of about 15 cm or less.

Aspect 44. The method of any of the above aspects, wherein the centrifuge has a rotor with a diameter of about 10 cm or less.

Aspect 45. The method of any of the above aspects, wherein the centrifuge has a rotor that, when in operation, defines an area whose longest dimension is about 15 cm or less.

Aspect 46. The method of any of the above aspects, wherein the centrifuge has a rotor that, when in operation, defines an area whose longest dimension is about 10 cm or less.

Aspect 47. A method comprising: centrifuging a blood sample in a vessel for a period of time; varying the centrifugation speed to establish a linear compaction curve for at least one formed blood component over the period of time until compaction is complete; monitoring a centrifugation speed profile for at least a portion of the time period; and determining a sedimentation rate of the blood component based on the centrifugation speed profile.

Aspect 48. A method comprising: centrifuging a blood sample in a vessel for a period of time; collecting at least a first single image of the position of the interface between formed blood components and plasma at an initial time; collecting at least a second single image of the position of the interface between formed blood components and plasma at a second time when the sedimentation rate is still linear; and calculating the sedimentation rate of at least one formed blood component in the blood sample based on the calculated linear sedimentation rate and the hematocrit correction factor.

Aspect 49. A device for use with a sample, the device comprising:

A centrifuge having a centrifuge vessel holder configured to allow detection of blood component interface locations in the vessel holder during centrifugation.

Aspect 50. The device of aspect 49, wherein the centrifuge has a window to allow visual observation of the centrifuge vessel holder during centrifugation.

Aspect 51. The device of aspect 49, wherein the centrifuge has an illumination source to allow detection of the location of an interface of blood components in the sample.

Aspect 52. A system comprising: a centrifuge having a centrifuge vessel holder, wherein the centrifuge vessel holder is configured to allow detection of an interface position of blood components in the vessel holder during centrifugation; a sample handling system for transporting a blood sample from a first location to a location on the centrifuge; and a processor programmed to record the interface position during at least a portion of the centrifugation.

This document contains material that is subject to copyright protection. The copyright owner (the applicant herein) has no objection to the facsimile reproduction by anyone of the patent document and disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. The following notice shall apply: Copyright 2013-2014 Theranos, Inc.

While the above is a complete description of various embodiments of the present invention, various alternatives, modifications, and equivalents may be used. Therefore, the scope of the present invention should not be determined by reference to the above description, but rather by reference to the appended claims, together with their full range of equivalents. Any feature, whether preferred or not, may be combined with any other feature, whether preferred or not. The appended claims should not be interpreted as including a device-plus-function limitation unless such a limitation is explicitly stated in a given claim using the phrase "means for...". It should be understood that, as used herein and throughout the claims that follow, the meanings of "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Furthermore, as used herein and throughout the claims that follow, the meaning of "among" includes "among" and "above," unless the context clearly dictates otherwise. Finally, as used herein and throughout the claims that follow, the meanings of "and" and "or" include both conjunctive and disjunctive and may be used interchangeably unless the context clearly dictates otherwise. Thus, in contexts where the terms "and" and "or" are used, use of such conjunctions does not exclude the meaning of "and/or" unless the context clearly dictates otherwise.

Claims (15)

1. A method for determining the sedimentation rate of at least one formed blood component, comprising: The blood sample in the vessel was subjected to an accelerated blood component separation technique for at least a certain period of time; Capture at least a first single image of the location of the interface between the formed blood components and plasma in the vessel at an initial time; Capture at least a second single image of the location of the interface between the formed blood components and plasma during a second time period within the linear sedimentation time period; The sedimentation rate of at least one formed blood component in the blood sample is calculated based on the calculated linear sedimentation rate and the hematocrit correction factor. The accelerated blood component separation technology includes centrifugal separation; The accelerated blood component separation technology generates a first G-force for a first time period, followed by a larger second G-force for a second time period; the first G-force is sufficient to accelerate sedimentation but not so fast as to fully compact the formed components before sedimentation changes are observed within a linear range; wherein the second G-force is a high-speed rotation at least twice or more of the lower speed of the measured rotation. 2. The method of claim 1, further comprising calibrating the sedimentation rate according to the centrifuge-based technique using the sedimentation rate according to the reference technique. 3. The method of claim 2, wherein the reference technique is the automated Westergren technique. 4. The method of claim 3, wherein the reference technique is the artificial Westergren technique. 5. The method of claim 1, wherein the blood sample is 25 µL or less. 6. The method of claim 1, wherein centrifugal separation occurs at a first speed for a first time period, and then occurs at a faster second speed for a second time period. 7. The method of claim 6, wherein the first velocity is configured to provide a substantially consistent force to the sample, and the second velocity is configured to provide a greater force but within a larger range of force variation relative to the substantially consistent force associated with the first velocity. 8. The method of claim 6, wherein the first G-force is applied to the sample with a substantially uniform force, and the second G-force is applied with a force that is larger than the first G-force but also within a larger range of force variation. 9. The method of claim 8, wherein the first G force is in the range of 35 G to 45 G. 10. The method of claim 8, wherein the first G force is in the range of 30 G to 50 G. 11. The method of claim 8, wherein the first G force is in the range of 10 G to 60 G. 12. The method of claim 8, wherein the second G force is in the range of 10 G to 100 G. 13. The method of claim 8, wherein the first G-force is sufficient to accelerate settlement but not so fast as to fully compact the forming component before settlement change becomes apparent when the settlement change is within a linear range. 14. The method of claim 8, wherein the second G-force has a high-speed rotation that is at least twice or higher than the measured rotation at a lower speed. 15. A method for determining the sedimentation rate of blood components, comprising: The blood sample in the container is centrifuged for a period of time; The centrifugation speed is varied to establish a linear compaction curve for at least one shaped blood component within the stated time period until compaction is complete; Monitoring the centrifugal separation rate distribution map for at least a portion of the time period; and The sedimentation rate of blood components is determined based on the centrifugation velocity distribution map. The centrifugation occurs with a first G-force for a first time period, followed by a larger second G-force for a second time period; the first G-force is sufficient to accelerate sedimentation but not so fast as to fully compact the forming component before sedimentation changes are observed when sedimentation changes are within a linear range; wherein the second G-force is a high-speed rotation that is at least twice or more the speed of the measured rotation at the lower speed.