Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
  • B01D21/30—Control equipment
  • B01D21/32—Density control of clear liquid or sediment, e.g. optical control ; Control of physical properties
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
  • B01D21/26—Separation of sediment aided by centrifugal force or centripetal force
  • B01D21/262—Separation of sediment aided by centrifugal force or centripetal force by using a centrifuge
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
  • B01D21/30—Control equipment
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/04—Investigating sedimentation of particle suspensions
  • G01N15/05—Investigating sedimentation of particle suspensions in blood
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/49—Blood
  • G01N33/491—Blood by separating the blood components
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5021—Test tubes specially adapted for centrifugation purposes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/04—Investigating sedimentation of particle suspensions
  • G01N15/05—Investigating sedimentation of particle suspensions in blood
  • G01N2015/055—Investigating sedimentation of particle suspensions in blood for hematocrite determination

Definitions

• ESR Erythrocyte sedimentation rate
• a sedimentation rate also called a sedimentation rate or Biernacki Reaction
• Erythrocyte sedimentation rate is the rate at which red blood cells sediment, typically measured over a period of one (1) hour. It is a common hematology test and is a non-specific measure of inflammation.
• anti-coaguiated 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 reported in mm/hour.
• the Westergren method requires collecting 2 ml of venous blood into a tube containing 0,5 ml of sodium citrate. The sample should be stored no longer than 2 hours at room temperature or 6 hours at 4 °C.
• the blood is drawn into the Westergren-Katz tube to the 200 mm mark.
• the tube is placed in a rack in a strictly vertical position for one hour at room temperature, at which time the distance from the lowest point of the surface meniscus to the interface between red-cell free plasma and the portion of the sample occupied by red-cells measured.
• the distance moved by the erythrocyte interface expressed as millimeters in 1 hour (mm/h) is the ESR.
• the ESR is governed by the balance between pro-sedimentation factors, mainly fibrinogen (but possibly also the levels of serum C-reactive protein (CRP), immunoglobulins A. and G, alphaf I )-acid- glycoprotein and afpha(l)-antitrypsin), and sedimentation resisting factors, mainly the negative charge of the erythrocytes (zeta potential).
• pro-sedimentation factors mainly fibrinogen (but possibly also the levels of serum C-reactive protein (CRP), immunoglobulins A. and G, alphaf I )-acid- glycoprotein and afpha(l)-antitrypsin), and sedimentation resisting factors, mainly the negative charge of the erythrocytes (zeta potential).
• fibrinogen but possibly also the levels of serum C-reactive protein (CRP), immunoglobulins A. and G, alphaf I )-acid- glycoprotein and afpha(l)-antitrypsin
• lymphoproiiferaiive disorders in which one or more immunoglobulins are found in high concentrations.
• Rouleaux formation can, however, be a normal physiological finding in horses, cats, and pigs.
• ESR is increased by any cause or focus of inflammation. ESR is increased in pregnancy and rheumatoid arthritis, and decreased in polycythemia, sickle cell anemia, hereditary spherocytosis, and congestive heart failure. The basal ESR is slightly higher in females.
• ESR ESR-Reacttve Protein
• sedimentation rate test It may be desirable to have sedimentation rate test that can be completed in a very short time, such as but not limited to being on the order of seconds to a few minutes.
• sedimentation rate measurements that use only small blood volumes, such as can be obtained by altemaie site, non- venous blood draws or minimal venous draws. It may be further desirable to make the sedimentation measurement in an automated fashion (no human observation required) and to create an objective record of the measurement. Additionally, further information useful in optimizing management of patients may be obtained by performing and/or maximizing the speed of multiplexed measurement of other analytical parameters in parallel with sedimentation rate measurement.
• the sedimentation rate measurement method may use (1) centrifugal techniques for separating red blood cells from plasma and (2) video and/or still imaging capability. Both may be used alone or in combination to accelerate erythrocyte sedimentation and to measure its rate. Of course, techniques other than
• centrifugation for accelerating sedimentation may be used in place of or in combination with centrifugation to separate blood components.
• the method may advantageously enable (I) rapid measurement of ESR (seconds) with small blood sample volumes such as about 20-25 microliters ("uL” or " ⁇ ,") or less, (2) use of automated image analysis to determine both red blood cell sedimentation rate and hematocrit, and/or (3) automated techniques to compensate for
• Westergren techniques can be used on sample with fibrinogen and/or hematocrit levels outside the narrow range required by Westergren testing.
• corrected ESR can be acquired in a matter of seconds using a small blood v olume and which compensates for effects of hematocrii ESR.
• the results acquired in a matter of seconds during initial centrifugation can accelerate deliver of a diagnosis to the patient.
• a common preprocessing step already involves separating red and white ceils from plasma or serum prior to measurements of cellular markers and of anaiytes present in plasma/serum.
• an ESR measurement along with such a pre-processing procedure that will already be performed during the course of assay preparation.
• the ESR measurement will not create significant burden in terms of additional processing time or use of limited quantities of blood available from non-venous collection methods.
• assay processing including pre-processing step(s), may occur in a single instrumented system.
• some embodiments may perform one or more steps in one instrument and another one or more steps in another instrument.
• a typical protocol may take 20 uL of blood in a centrifuge vessel and spin in a swing-out centrifuge rotor at 4000 rpm (58G*g) for about 10 s. During this time, the interface between the portion of the sample containing the red blood cells and that cleared of red blood cells is observed by video imaging. Although other time periods are not excluded, it can be advantageous to obtain the ESR measurement in this short period of time. 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 for measurement of raw ESR.
• the spin speed is increased to pack the red blood cells.
• Hematocrit is determined by image analysis of the packed red blood cells and the supernatant plasma volumes.
• other techniques for measuring hematocrit may also be used to correct "raw" ESR values.
• At least one of the embodiments herein may have ESR corrected without using calculations of the slope of an essentially linear transform of the non-linear (exponential) portion of the sedimentation curve.
• At least one of the embodiments herein may have ESR corrected without calculating a mathematical function for a plurality of the erythrocyte/plasma interface positions occurring in a non-linear portion of the sedimentation curve.
• At least one of the embodiments herein may have ESR corrected without selecting a segment of the sedimentation curve which lies in said non-linear portion of the sedimentation curve.
• At least one of the embodiments herein may have ESR corrected based only on measurements of linear portion(s) of the sedimentation curve.
• At least one of the embodiments herein may have ESR corrected based on measurements which consists essentially of linear portion(s) of the sedimentation curve.
• consists essentially of we mean at least 90% or more of the measurement is based on the linear portion(s).
• At least one of the embodiments herein may have ESR. corrected without determining a mathematical function for a non-linear segment of the sedimentation curve representative of the magnitude of intercellular erythrocyte repulsion in the blood sample.
• At least one of the embodiments herein may have ESR corrected without negating the time period during the centrifugation of the sample during which a linear portion of the sedimentation curve is formed.
• At least one of the embodiments herein may have ESR corrected for hematocrit using hematocrit measurements not derived from centrifugal techniques, such as for example, lysis of red cells with detergent and mixing with ferricyanide and cyanide followed by measurement of the absorbance of the cyan-met-hemoglobin fonned.
• At least one of ihe embodiments herein may have the blood sample adjusted so that it is at a known hematocrit level for the sedimentation measurement.
• a method comprising: using an accelerated blood component separation technique on a blood sample for a period of time to separate formed blood components from plasma; determining a sedimentation rate of the formed blood component based on at least the following: a time-related compaction curve and a hematocrit correction factor, wherein the time-related compaction curve for at least one formed blood component in said blood sample is determined after accelerated blood component separation has begun, said compaction curve having an initial approximately linear portion and a non-linear portion after the linear portion.
• a method comprising: centrifuging a blood sample in a vessel for a period of time; establishing a time -related compaction curve for at least one formed blood component in said blood sample after centrifuging has begun, said compaction curve having an initial approximately linear portion; correcting for hematocrit effect on sedimentation rate of the formed blood component by using a hematocrit correction factor on the approximately linear portion of said compaction curve.
• the method comprises calibrating sedimentation rates from centrifuge based technique with sedimentation rates from a reference technique.
• the reference technique is the Westergren technique.
• the sample is about 25 uL or less.
• centrifuging occurs at a first speed for a first period of time and then at a second, faster speed for a second period of time.
• centrifuging comprises using a centrifuge configured to allow the blood sample to be visually observed during centrifugation to establish interface positions of one or more formed blood components in the blood sample.
• centrifuging comprises using a centrifuge having a window thereon to enable visual observation of the blood sample to establish erythrocyte/plasnia interface positions over time.
• centrifuging comprises using a centrifuge, a light source, and an image capture device to enable visual observation of the blood sample to establish formed blood component/plasma interface positions over time.
• compaction curve data is collected by capturing a plurality of images of interface positions of one or more formed blood components in a centrifuge vessel over the time period.
• pixel positions in the plurality of images are used to accurately determine interface position.
• compaction curve data is collected by capturing a single image of an interface position of one or more formed blood components in the centrifuge vessel after a time period, wherein sedimentation rate is calculated based on position of the meniscus of the supernatant liquid and the interface position.
• compaction curve data is collected while the sample is being centrifuged.
• centrifugation is used to obtain hematocrit
• correcting for hematocrit comprises calculating a mathematical function for a plurality of formed blood component interface positions occurring in said curve, said function being operative to correct for sedimentation rate variations due to hematocrit.
• hematocrit measurement in the sample is derived from a technique separate from centrifugation
• image transformation is used for conversion of a curved interface to a flat interface.
• image transformation parameters are selected, video of formed blood component interface position is put through image transformation, and then a region of interest is chosen that covers both the whole range of positions for both air/plasma interface and erythrocyte interface.
• each timepoint in the video pixel intensity values for each row across a sample vessel containing the sample, within the region of interest are averaged to produce a single column representing the intensity radially down the sample vessel.
• a linear region of a sedimentation profile is used to extract a sedimentation rate.
• the formed blood component is white blood cells.
• the formed blood component is platelets.
• the method comprises performing image transformation on said images to transform images with curved interfaces into corrected images with straight line interfaces; establishing a time-related compaction curve based on interface positions in said corrected images, for at least one formed blood component in said blood sample after centrifuging has begun.
• the method comprises: using a programmable processor-controlled system to transfer at least a portion of a blood sample from a blood sample location into a centrifugation vessel; using a sample handling system under programmable processor control to transfer said vessel from a first addressable position to a centrifuge with a second addressable position; centrifuging the blood sample in the vessel for a period of time; collecting at least one image of formed blood component and plasma interface position after centrifuging; establishing a time -related compaction curve based on interface position(s) in the image, for at least one formed blood component in said blood sample after centrifuging has begun.
• the vessel is removed from the centrifuge to obtain said image.
• the vessel is returned to the centrifuge after said image is obtained.
• the method comprises varying centrifuging speed to establishing a linear compaction curve of at least one formed blood component over the period of time until compacting has completed; monitoring centrifuging speed profile for at least a portion of the time period; and determining blood component sedimentation rate based on the centrifuging speed profile.
• the method comprises collecting at least a first single image of formed blood component and plasma interface positions at an initial time; collecting at least a second single image of formed blood component and plasma interface positions at a second time while rate of sedimentation is still linear; calculating sedimentation rate for at least one formed blood component in said blood sample based on linear sedimentation rate calculated and a hematocrit correction factor.
• a method comprising: centrifuging a blood sample in a vessel for a period of time; using imaging of the vessel in a single state condition to establish sedimentation rate; and correcting for hematocrit effect on sedimentation rate of the formed blood component by using a hematocri t correction factor.
• using imaging can comprise using a single image of the vessel to determine sedimentation rate.
• a meniscus of supernatant liquid in the vessel shows an initial level and an interface position of formed components with the supernatant liquid shows current position, from which
• sedimentation rate is calculated.
• some embodiment may use a plurality of images for sedimentation rate calculation, but all of the images are of the vessel while the vessel is in a single state condition.
• all of the images are of the vessel at a single point in time.
• all of the images are of the vessel while the formed component interface position is not changing in the vessel.
• the dual speed is used to extend the dynamic range of the ESR assay.
• some embodiments may use centrifugation at three or more speeds.
• centrifuge speed may be viewed as a proxy for 0 force applied to the sample, and it should be understood that the concept herein can be applied to other embodiments that provide for accelerated sedimentation of formed samples but do not use a centrifuge.
• a method comprising using an accelerated blood component separation technique on a blood sample in a vessel for at least one period of time; capturing at least a first single image of formed blood component and plasma interface positions in the vessel at an initial time; capturing at least a second single image of formed blood component and plasma interface positions at a second time within a linear sedimentation time period; and calculating a sedimentation rate for at least one formed blood component in said blood sample based on linear sedimentation rate calculated and a hematocrit correction factor.
• the accelerated blood component separation technique comprises centrifugation.
• the method further comprises calibrating sedimentation rates from centrifuge-based technique with sedimentation rates from a reference technique.
• the reference technique is an automated
• the reference technique is a manual Westergren technique.
• the blood sample is about 25 ⁇ _, or less.
• centrifuging occurs at a first speed for a first period of time and then at a second, faster speed for a second period of time.
• the first speed is configured to provide a substantially consistent force on the sample, while the second speed is configured to provide greater force but at a greater range of force variability relative to the substantially consistent force associated with the first speed.
• the accelerated blood component separation technique occurs at a first G-force for a first period of time and then at a second, greater G-force for a second period of time.
• the first G-force is provided at a substantially consistent force on the sample, while the second first G-force is provided at greater force but also at a greater range of feree variability relative to the substantially consistent force of the first G-force.
• the first G-force is in the range of about 35G to about 45G,
• the first G-force is in the range of about 30G to about 50G.
• the first G-force is in the range of about 10G to about 60G.
• the second G-force is in the range of about 10G to about 1G0G.
• the first G-force is one that is sufficient accelerate sedimentation but not so fast that the formed components become fully compacted before the change in sedimentation is visualized while in the linear range.
• the second G-force has a high speed spin that is at least about 2 times or more of the lower speed, measurement spin.
• a method comprising using force on a blood sample to reduce measurement time associated with calculating a sedimentation rate for at least one formed blood component in said blood sample
• a device for use with a sample, the device comprising: a centrifuge having a centrifuge vessel holder configured to allow for detection of blood component interface position in the vessel holder during eentrifugation.
• the centrifuge has window to allo w for visual observation of the centrifuge vessel holder during eentrifugation.
• the centrifuge has an illumination source to allow for detection of blood component interface position in the sample.
• a system comprising a centrifuge having a centrifuge vessel holder configured to allow for detection of blood component interface position in the vessel holder in the vessel holder during eentrifugation; 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 interface position during a least a portion of eentrifugation.
• a method comprising at least one technical feature from any of the other embodiments herein.
• a method may comprise at lea st any two technical features from any of the other embodiments herein.
• a device may comprise at least one iechnical feature from any of the other embodiments herein.
• a device may comprise at least any two technical features from any of the other embodiments herein.
• a system may comprise at least one technical feature from any of the other embodiments herein.
• a system may comprise at least any two iechnical feaiures from any of the other embodiments herein.
• Figure 1 shows a graph of red cell sedimentation of high, medium, and low ESR blood samples in Westergen-Katz tubes.
• Figures 2A-2B are images of blood samples in transparent centrifugation vessels.
• Figure 3 shows a schematic of a centrifuge with one embodiment of a detection system.
• Figures 4-5 show images captured using one embodiment of a detection system.
• Figures 6A-7C sho a series of corrected and uncorrected images of interfaces in a blood sample undergoing centrifugation.
• Figures 8A-8B show various kymographs for one test sample.
• Figure 9 shows a sedimentation graph for one test sample
• Figures I OA- 10B show sedimentation graphs with various fitted functions fitted to the claia of Figure 9 plotted thereon,
• Figures 1 1-14 are graphs showing various sample sedimentation characteristics for samples with various levels of added fibrinogen.
• Figure 15 shows sedimentation rates for several blood samples manipulated to have different hematocrit levels
• Figure 16 is a graph of interface positions over time for the samples with different hematocrit levels also shown in Figure 15.
• Figure 17 is a graph of interface positions over a 10 second period of time for one sample with different hematocrit levels
• Figure 18A shows an ESR graph of one embodiment herein without hematocrit correction.
• Figure 18B shows an ESR graph of one embodiment herein with hematocrit correct on.
• Figure 18C shows a graph of hematocrit measurement based on hemoglobin concentration according to one embodiment herein.
• Figures 19 and 20 illustrate sedimentation rates for several samples (as specified in Figure 15) plotted using non-LOG and LOG axis.
• Figure 21 shows a kymograph illustrating a white blood cell interface.
• Figure 22 shows a schematic of one embodiment of an integrated system having sample handling, pre-processing, and analysis components.
• Figure 1 the kinetics of erythrocyte/plasma interface for a set of blood samples is shown.
• Figure 1 shows the kinetics of erythrocyte sedimentation in
• a variety of techniques may be used to establish a sedimentation rate curve for one or more formed blood components. Although the present application is described mostly in the context of measuring erythrocyte sedimentation rate, systems and methods herein can also be adapted for use in measuring sedimentation rates for other formed blood components such as but not limited to white blood cells, platelets, or the like.
• one technique described herein comprises taking images at several time points during sedimentation by placing the sample vessel in a centrifuge, spinning for a few seconds, stopping the spin, removing the vessel, placing it in a viewer, taking an image, and repeating the above to obtain multiple images over time. From a device simplicity standpoint, it is helpful in that it simplifies hardware implementation for obtaining such images.
• the ability to measure sedimentation is discussed elsewhere herein where the slope from the initial (linear) part of the sedimentation curve is used to calculate the ESR.
• the centrifuge vessel may be made in whole or in part of transparent material such as transparent plastic (injection-molded polystyrene).
• the transparent portions may be windows, clear ports, or clear strips in the vessel aligned to allow for imaging of the desired blood component interface of sample in the vessel.
• the radius of the centrifuge vessel at its mid-point is 35 mm (radial distance from the axis of rotation). In one embodiment, outer radius is 35 mm, inner (i.e.
• centrifuge designs and features including dimensions for the centrifuge vessels, construction of the centrifuge rotor, and centrifuge size are disclosed in copending U.S. patent applications Ser. Nos. 13/355,458 and 13/244,947, all fully incorporated herein by reference for all purposes.
• Other components of the present system including suitable imaging devices and fluid handling systems are also described in the applications incorporated by reference.
• the ability of digital cameras such as those described in those applications may 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 cells and plasma.
• only two measurements taken at early times (seconds) after centrifugation has begun is sufficient to define the sedimentation rate with high precision.
• some may take the first image after an initial minimum centrifuge speed is reached and then a second image may be taken about 10 seconds later.
• other time periods for the images are not excluded so long as they are in the linear portion of the sedimentation curve.
• FIG. 2A and 2B Viewing Figures 2A and 2B, erythrocyte interface position in a stationary (vertical) tube (short time of centrifugation) can be seen.
• the swinging centrifugation vessel is stopped and oriented vertically in the Figures 2.
• a and 2B Of course, stationary imaging does not need tube to be vertical (so long as it is done quickly) since surface tension holds interface in place. Typically, this is done within a second or two else RBC interface will begin to flow.
• Some embodiments may magnify the image so that more pixels are associated with the interface and thus more pixels are associated with change in position of the interface. Some may use detectors with greater numbers of pixels per unit area. This increases the sensitivity of the measurement by measuring more pixels and having the ability to detect even more subtle changes in interface position.
• a method which uses a transparent window in the centrifuge housing so that a video record of sedimentation can be made during the lo -speed centrifugation. Moreover, the centrifugal field causes the meniscus to become straighter (at right angles to the centrifugal force vec tor) making measurement of small settling distances easier. This may be particularly true when images are captured while the centrifuge rotor is spinning. By spinning a small volume (20-25 uL) of blood at intermediate speeds (typically 4000 rpm, although 1000 to 6000 rpm may also suitable), almost complete sedimentation of red blood cells is achieved in this embodiment within about three minutes.
• one method may take sedimentation measurements for a few seconds at relatively low speed ( 1000 to 4000 rpm) then the speed would be increased to about 10,000 rpm for about three minutes to pack the red blood cells and determine the hematocrit.
• the low speed spin is about 40G.
• some embodiments may have the spin create G forces in the range of about 40G to 100G.
• some embodiments may have the spin create G forces in the range of about 10G to 60G.
• some embodiments may have the spin create G forces in the range of about 10G to 100G.
• the desired G speed is one that is sufficient accelerate sedimentation but not so fast that the formed components become fully compacted before the change in sedimentation is visualized while in the linear range.
• this mufti-stage spinning at different centrifugation forces allows for imaging for sedimentation and then rapid spin down to achieve compaction of blood components and separation from blood plasma.
• some embodiments may have the low speed spin in the l OOOrpm +/- 20% rpm range.
• some embodiments may have the low speed spin in the 800rpm to 1500 rpm range.
• some embodiments may have a high speed spin that is about 2 times or more of the lower speed, measurement spin.
• some embodiments may have a high speed spin that is about 3x or more of the lower speed, measurement spin.
• some embodiments may have a high speed spin that is about 4x or more of the lower speed, measurement spin.
• some embodiments may have a high speed spin that is about 5x or more of the lower speed, measurement spin.
• some embodiments may have a high speed spin that is about 6x or more of the lower speed, measurement spin.
• some embodiments may have a high speed spin that is about 7x or more of the lower speed, measurement spin.
• some embodiments may have a high speed spin that is about 8x or more of the lower speed, measurement spin.
• some embodiments may have a high speed spin that is about 9x or more of the lower speed, measurement spin.
• some embodiments may have a high speed spin that is about 1 Ox or more of the lower speed, measurement spin.
• some embodiments may have a high speed spin that is about 15x or more of the lower speed, measurement spin.
• some embodiments may have a high speed spin that is about 20x or more of the lower speed, measurement spin.
• a controller or other device can be used to maintain the desired rpm so that the desired formed component measurement can be made.
• This desired rpm can be at a controlled rate +/- 1 % of the target RPM.
• the controlled rate may be +/- 2% of the target RPM.
• the controlled rate may be +/- 3% of the target RPM.
• the controlled rate may be +/- 4% of the target RPM.
• the controlled rate may be +/- 5% of the target RPM.
• the process may involve a period of low-speed centrifuge spin at a controlled rate and then a high speed spin where the primary factor is having the centrifuge disc spin above a minimum threshold, wherein exact RPM is less important versus maintaining at least a minimum spin rate.
• an image capture device 110 may be located near the centrifuge 100, with a light source 1 12 such as but not limited to a green, LED positioned to provide illumination from an opposing location.
• a light source 1 12 such as but not limited to a green, LED positioned to provide illumination from an opposing location.
• the image capture device 1 10 may be a still camera, a high speed camera, a video camera, or other device sufficient to detect the location of the interface.
• other detectors such as but not limited to non-image capture devices are also not excluded.
• one non-visual imaging device here may be a photodiode which can function as a detector to detect when a blood component interface passes the detector, or by bulk transmission of light to detect proportion of volume blocked by RBCs or other blood component(s).
• Non-visual imaging detectors can be used even if they may not actually convey a visual image but can still detect interface level position and/or position change in the sample.
• the image capture device 1 10 may be a digital camera.
• Image capture devices may also include charge coupled devices (CCDs) or photomultipliers and phototubes, or photodetector or other detection device such as a scanning microscope, whether back-lit or forward- lit.
• CCDs charge coupled devices
• CMOS complementary metal-oxide-semiconductor
• CMOS complementary metal-oxide-semiconductor
• CCDs charge coupled devices
• imaging devices may employ 2-d imaging, 3-d imaging, and/or 4-d imaging (incorporating changes o ver time). Imaging devices may capture static images. The static images may be captured at one or more point in time. The imaging devices may also capture video and/or dynamic images. The video images may be captured continuously over one or more periods of time. Any other description of imaging devices and/or def ection units may also be applied, preferably so long as they are able to detect changes in interface position.
• a light source 1 12 may be a light-emitting diode (LED) (e.g., gallium arsenide (GaAs) LED, aluminum gallium arsenide (AlGaAs) LED, gallium arsenide phosphide (GaAsP) LED, aluminum gallium indium phosphide (AlGalnP) LED, galiium(ni) phosphide (GaP) LED, indium gallium nitride (InGaN) / gailium(iiJ) nitride (Ga ) LED, or aluminum gallium phosphide (AlGaP) LED).
• LED light-emitting diode
• GaAs gallium arsenide
• AlGaAs aluminum gallium arsenide
• GaAsP gallium arsenide phosphide
• AlGalnP aluminum gallium indium phosphide
• GaP galiium(ni)
• a light source can be a laser, for example a vertical cavity surface emitting laser (VCSEL) or other suitable light emitter such as an Indium-GaUium-Aluminum-Phosphide (InGaAiP) laser, a Gallium-Arsenic Phosphide/Gallium Phosphide (GaAsP/GaP) laser, or a Gallium-Aluminum- Arsenide/Gallium- Aluminum-Arsenide (GaAIAs/GaAs) laser.
• VCSEL vertical cavity surface emitting laser
• GaAsP/GaP Gallium-Arsenic Phosphide/Gallium Phosphide
• GaAIAs/GaAs Gallium-Aluminum- Arsenide/Gallium- Aluminum-Arsenide
• light sources may include but are not limited to electron stimulated light sources (e.g., Cathodoluminescence, Electron Stimulated Luminescence (ESL light bulbs), Cathode ray tube (CRT monitor). Nixie tube), incandescent light sources (e.g., Carbon button lamp, Conventional incandescent light bulbs, Halogen lamps, Globar, Nernst lamp), electroluminescent (EL) light sources (e.g., Light-emitting diodes - Organic light-emitting diodes. Polymer light- emitting diodes, Solid-state lighting, LED lamp, Electroluminescent sheets Electroluminescent wires), gas discharge light sources (e.g...
• electron stimulated light sources e.g., Cathodoluminescence, Electron Stimulated Luminescence (ESL light bulbs), Cathode ray tube (CRT monitor).
• Nixie tube incandescent light sources (e.g., Carbon button lamp, Conventional incandescent light bulbs,
• Fluorescent lamps Inductive lighting, Hollow cathode lamp, 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, Hydrargyrum medium-arc iodide lamps. Mercury-vapor lamps, Metal halide lamps, Sodium vapor lamps, Xenon arc lamps).
• a light source may be a bioluminescent, chemiluminescent, phosphorescent, or fluorescent light source.
• a centrifuge vessel 1 14 containing a blood sample therein may be positioned so as to be between the image capture device 1 10 and the light source 1 12 to enable the position of the formed blood component interface(s) in the vessel to be visualized.
• the centrifuge rotor 1 16 may be configured to have an opening, a window, or other area that allows the centrifuge vessel 1 14 to be visualized during centtifugation. Measuring sedimentation during centrifugation spin may be used, but it should be understood that measuring
• the axis of rotation of the centrifuge rotor 1 16 may be vertical It should be understood that other axis of rotation such as horizontal or angled axis of rotation are not excluded. Some embodimenis may have a first orientation during one time period and a different orientation during a second or other time period.
• the positions of the top and/or bottom of the centrifuge vessel are obtained by imaging as reference points, and later these are used to calibrate the liquid and interface levels.
• Figure 4 shows a camera view of a centrifuge vessel 1 14 in the centrifuge. Illumination from light source 1 12 from behind the centrifuge vessel 1 14 allows for visualization of blood sample S and the blood/air interface 120. The direction of rotation is shown by arrow 122.
• Figure 5 is an image of sedimenting red blood cells during centrifugation. Air/plasma interface 130 and plasma/red blood cell interface 132 are clearly discernable as sharp lines (separating spatial regions of different contrast) in the image. Space 134 above the air/plasma interface, plasma 136, and light 138 blocked red blood cells are also shown in the image of Figure 5.
• strobe illumination or capture frames synchronized to the rotor position are not excluded, but in the present embodiment are not required for image capture.
• a 200 ms exposure (short relative to spin times) for the CCD camera image acquisition makes the interfaces 130 and 132 clearly visible during the spin (see Figure 5). This was long compared to the rotation period (i.e. thai many rotations occur during the time, so that images blur out).
• the image blurs around the rotor axis so that the air/plasma interface and plasma/erythrocyte interface are visible as arcs (though there are maybe strobe effects that may also be taken into account).
• light transmitted through two regions of the centrifuge vessel 1 14 may include the air 134 above the liquid, and the plasma between the air/plasma and plasma/erythrocyte interfaces 130 and 132 as labeled.
• the air/plasma interface 130 itself is visible as an arc. Essentially no light makes it through the vessel 1 14 where the erythrocytes are (although it should be understood that this region is not completely dark because of the light transmitted when the vessel 1 14 rotates out of the blocking position).
• images are captured for three minutes at five frames per second, with long exposure ( -'200ms), then processed to extract the sedimentation curve.
• the rate of imaging includes but is not limited to 1, 2, 4, 8, 16, 32, 64, or 128 images per second.
• exposure time includes but is not limited to 10, 20, 40, 80, 160, 320, or 640 ms.
• Temperature during measurement may also be varied. Although many embodiments herein had measurements performed at room temperature, but other temperatures e.g. 37C are not excluded. Effect of temperature would be taken into account in the calibration, such as for determining empirical parameters of the correction factor. Also, time to centrifuge spin up was typically about 3 seconds, but faster or slower spin up times are not excluded.
• the sedimentation rate of the desired formed blood component being measured may be defined by:
• times of sedimentation are usually defined as starting following the rotor 1 16 reaching its target speed when the buckets holding the centrifuge vessels 1 14 are oriented radially in the spin plane and so are in optimal position for image capture and processing.
• one embodiment herein may use an image preprocessing step prior to analysis which 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 brings off-center pixels in line with the central axis in a way that has negligible effect on the y positions of the interfaces so that the blurred -out arcs from the rotating tube are now horizontal stripes,
• an initial image transform may be used to compensate for arcs.
• the image in Figure 6B shows a rectangle 150 with the selected area of interest across which the horizontal averaging is performed, and two short horizontal lines showing where the algorithm has identified the position of the air/plasma interface 30 and the plasma/erythroeyte interface 132.
• This image transformation is desirable to remove the effects of the vertical lines seen in Figure 6A, caused by a strobe effect between the frequency of rotation of the centrifuge and the acquisition frequency of the camera.
• a thin vertical (i.e. radial) section measurement would be vulnerable to these lines, which move slowly across the image, but the straightening transform allows averaging in the x-direetion (at right angles to the radial) and makes the profile immune to the effects of the moving lines. This procedure also improves the signal to noise ratio.
• Figures 7A-7C examples showing different degrees of arc compensation are shown. Selection of image transformation parameters can be chosen to introduce a desired level of correction.
• Figure 7 A shows compensation that is too little.
• Figure 7B shows compensation that is just right.
• Figure 7C shows too much arc compensation.
• the acquired image information which may be a video, is put through the transformation.
• a region of interest may be chosen that covers both the whole range of positions for both the air/plasma interface 130 and the erythrocyte interface 132.
• some embodiments may choose a region of interest covering only one of the interfaces 130 or 132.
• some embodiments may be configured to target one or more other areas of interest in the sample.
• one embodiment of the technique herein averages the pixel intensity values for each row (across the vessel 1 14) within the region of interest 350 to produce a single column representing the intensity radially down the vessel.
• the columns for each timepoint are then assembled into a kymograph, i.e. an image where the x-axis represents time and the y axis represents radial position along the tube.
• Figure 8A shows a kymograph according to one embodiment described herein.
• the kymograph of Figure 8A shows average image intensity down the tube (y-axis) o ver time (x-axis).
• Figure 8A shows an air interface 130, plasma 136, plasma/red blood cell interface 1 32, and red blood cells 40. 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 light transmitted through the plasma 1 36, and the dark area at the botto is where the light is blocked by the red blood cells 140.
• Figure 8B shows that, to extract the position of the air/plasma interface and the plasma/erythrocyte interface, a first derivati v e (edge detection) of the interface with respect to time may be determined. Derivative is with respect to distance down the tube (y-axis) and not time (x-axis).
• Figure 8B shows the positions of the air/plasma (upper) interface 130 and plasma/erythrocyte (lower) interface 132.
• the positions of the two local maxima of the image in Figure 8F3, one representing the air/plasma interface and other the plasma/erythrocyte interface are determined.
• the y-position of the top and bottom of the centrifuge tube (such as recorded from the stationary tube image shown in Figure 2) are used as reference locations together with knowledge of the shape of the centrifuge vessel.
• 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 Figure 9 is the result of one nonlimiting example of a centrifuge-based method of determining a sedimentation curve extracted from a video record.
• Figure 10A One such example is shown in Figure 10A.
• data in the graph is shown as black dots 200
• the x-axis is time in seconds
• the y-axis is the volume fraction occupied by red blood cells.
• a single exponential fit is shown as line 202.
• Figure 10B data in the graph is shown as black dots 200.
• Figure 10B shows a substantially bi-linear fit. The gradient of the initial linear portion shown by linear fit 210 may be determined, as well as the time 212 of the transition between the initial linear section, and the non-linear region where packing slows, shown here by the red line 214.
• the parameter of interest responds to the concentration of certain plasma proteins and can be directly affected 'manipulated by adding one of these proteins, (e.g., fibrinogen) to the blood sample.
• exogenous fibrinogen was used to create blood samples with ESR values spanning the whole range of interest (0— 120mm/h in the Westergren method).
• Figure 11 shows how adding fibrinogen increases the Westergren ESR values.
• each of these parameters can be used to obtain an estimate of the Westergren ESR value.
• the single exponential-fit time constant and the packing onset time both ha ve the advantage of being independent of y-seale.
• the packing onset time and the initial linear gradient have the advantage of having clear physical meanings.
• Figure 1 1 shows that Westergren ESR values increase with increasing added Fibrinogen.
• Figure 1 1 illustrates a single sample with different levels of fibrinogen added therein.
• Figure 12 shows a time constant from a single exponential fit of the raw sedimentation curve which shows good correlation with added fibrinogen levels
• Figure 13 shows time to the onset of ceil packing which shows good correlation with added fibrinogen levels.
• Figure 14 shows initial linear gradient of the raw sedimentation curves which show good correlation with added fibrinogen levels.
• hematocrit is another factor that affects Westergren and other ESR measurements.
• Westergren erythrocyte sedimentation is strongly affected by hematocrit.
• many laboratories either do not report results for samples with hematocrits greater than about 45 % or adjust the sample hematocrit to a fixed level (usually 45 %) before measuring ESR.
• the present embodiment of the method is actually better than the Westergren technique, in that Westergren saturates (i.e. does not respond to fibrinogen ⁇ I0mg/ml), whereas the present embodiment of the method does not saturate out to i5nig/nii.
• Centrifuge -based ESR sedimentation is even more strongly affected by hematocrit levels than measurements under gravity.
• the increased dependency on hematocrit is also because of the lower volumes - and consequently smaller vessel dimensions.
• Increasing hematocrit typically means the erythrocytes start closer together, increasing the viscosity of the blood by presenting physical barriers to free movement, and decreasing the maximum distance the interface can move before the cells become packed, ail of which decrease the ESR, independent of fibrinogen from inflammation.
• centrifuge-based ESR measurements performed by taking the same sample of blood and adjusting the hematocrit before measuring the ESR, show that a person with a typical hematocrit of 45% and a normal ESR of 22 mm/h would register as 5 rnm/h (very low) if the hematocrit were 60% and 93 mrn/h (very high) if the hematocrit were 35%, even though there are no changes in the plasma protein levels which are clinically important.
• variations of ESR due to hematocrit can dominate variations of ESR due to plasma proteins the clinician is interested in.
• FIG 16 shows complete sedimentation profiles while Figure 17 shows sedimentation profiles for a shorter period of time ( ⁇ 10s) for an initial measurement period for one sample adjusted to the given hematocrits.
• the sedimentation profiles for the samples show a sharp descent during the initial measurement period, with the interface position falling almost linearly with time during that initial measurement period. The sedimentation rate then slows as the red blood cells pack together.
• Many data sets corresponding to various hematocrits (as indicated) and various ESR rates are shown in Figure 16.
• Figure 18A shows the logarithm of the erythrocyte sedimentation rates extracted from sedimentation profiles uncorrected for hematocrit) .
• Figure 18A also shows that centrifuge- based sedimentation rates are strongly dependent on hematocrit, more so than the Westergren-based sedimentation rates.
• the narrow cross-section of the centrifuge tube increases hydrodynamic resistance to fluid flow due to red blood cells.
• the centrifugation process involves flow of plasma through a bed of red blood cells, which offer hydrodynamic resistance. This resistance is a function of the volume fraction of red blood cells, i.e., the hematocrit.
• the centrifuge-based sedimentation rates were corrected for effect of hematocrit.
• the correction used can be represented by,
• Uuncorr and Ucon- are the uncorrected (raw) and corrected sedimentation rates respectively
• ⁇ is the volume fraction of ceils (hematocrit)
• ⁇ TM* and ⁇ are empirical parameters obtained by curve fitting such as described below or as may be developed in the future.
• the correction factor represents a simple mathematical form to account for the increased drag exerted by red blood cells. It should be understood that this functional form was found to be able to correct for hematocrit, but other functions would work too.
• one way of calculating and ⁇ is by way of a calibration technique such as but not limited to the following: for a diverse set of samples (different hematocrits, ESR values, etc%), the ESR value is determined using a reference method, and by the centrifuge- based method.
• the tpmax and ⁇ parameters are determined as a calibration for each centrifuge setup and may change based at least in part on vessel geometry and volume of sample. Thus, if at least one of those factors is changed, it may be desirable to recalculate the parameters.
• the value for hematocrit may be known prior to the start of the centrifuge-based sedimentation test and in such situations, corrected ESR results can be obtained quickly based on the initial linear portion of the sedimentation and the known hematocrit level, without having to wait until the erythrocytes have been fully compacted by centrifugation.
• some embodiments may determine hematocrit levels during or after centrifugation.
• Hematocrit measurement by non-centrifugal before, during, or after centrifugation includes at least the following.
• One technique involves measurement of hemoglobin concentration. For example, in roughly 99% of the population, there is a 1 : 1 correlation between hemoglobin measurements and hematocrit le vels. Thus, if hemoglobin test data is available, the hematocrit level is generally already known before start of the centrifuge-based sedimentation test.
• FIG. 18C one embodiment of an assay protocol for hemoglobin- based hematocrit measurement will now be described.
• Blood was diluted 1 : 100 with water.
• the diluted sample was mixed (1 :3) with modified Drabkin's reagent (Sigma D5941, containing Contains sodium bicarbonate, potassium ferricyanide, and potassium cyanide supplemented with 0.015% Brij 35).
• modified Drabkin's reagent Sigma D5941, containing Contains sodium bicarbonate, potassium ferricyanide, and potassium cyanide supplemented with 0.015% Brij 35.
• the absorbance of the reaction product (Cyan-met-hemoglobin) was measured at 540 nm.
• the assay was calibrated with bovine hemoglobin (Sigma 2500) which gave a linear dose-response over the range 0 - 20 g/dL.
• Hematocrit can also be measured in the devices using a cuvette with a fixed depth and a blood sample diluted to a known extent. Description of a system with such a cuvette can be found in U.S. Patent Application Ser. NO. 13/244,947 fully incorporated herein by reference for all purposes. Hematocrit can be determined by microscopic measurement of the ( 1) the red cell count per field of view and (2) the average red cell volume. Favored methods are: (1) Dark field microscopy and (2) (1) + Fluorescence microscopy using fluorescently-labeled anti-human CD- 35 (red cell surface antigen). Image analysis techniques are then applied.
• one method of measuring hematocrit may involve measuring optical density of the sample. See for example Lipowsky et ai. "Hematocrit determination in small bore tubes from optical density measurements under white light illumination" Microvascular Research, Volume 20, Issue 1, July 1980, Pages 51 -70; http://dx.doi.Org/l 0.1016/0026- 2862(80)90019-9, fully incorporated herein by reference for ail purposes.
• Lipowsky discusses the relationship between the hematocrit of blood flowing in small bore glass tubes and its optical density (OD) under white light (tungsten) illumination has been examined for various tube luminal diameters. In at least some embodiments herein, all this data is available since a smallbore tube of blood is being illuminated.
• hematocrit le vel can be determined by testing a portion of the blood sample under microscopy or other magnified observation, such as but not limited to measuring the number and mean size of red blood cells in a defined observation area which may have a known size. In this manner, the hematocrit may be determined based on such visual characterization of the red blood cells.
• hematocrit level can be measured based on a completed centrifugation of the blood sample which compacts the red blood cells. This compacted level can be used to determine hematocrit.
• only linear portions of the centrifuge based sedimentation test are used to determine a corrected ESR.
• the first initial portion of interface position measurement which is linear, along with the final end portion which is also linear, are two portions of the sedimentation which may be used to calculate an ESR corrected for hematocrit.
• this non- limiting example may use a linear portion 1 82 of the sedimentation curve corresponding to an initial period after centrifugation and another linear portion 184 of the sedimentation curve near the end when compaction is essentially complete curve is substantial flat.
• the non-linear portion of the sedimentation curve therebetween linear portions 1 82. and 184 are substantially not used to calculate the hematocrit correction factor.
• a vessel containing the sample may be centrifuged under controlled conditions for a first period of time, such as but not limited to time period associated with the linear portion of the sedimentation curve.
• the centrifugation is controlled to a specific force range (such as an rpm range for the rotor of the centrifuge) such that there is a substantially consistent force applied to the accelerated sedimentation process.
• the sedimentation profile during the initial period is generally linear, and it may be desirable to capture the sedimentation image of formed components in the material after centrifugation during the initial linear period.
• the image can be taken in several scenarios such as but not limited to: a) while the vessel is in the centrifuge, b) when the vessel is in the centrifuge but the centrifuge is stopped, or c) by removing the vessel from the centrifuged and imaging it, in some embodiments, the sedimentation can be measured with a single picture.
• the level at the start time tO is the meniscus level of the supernate or the remaining solution abo ve the settled formed components.
• the level of sedimentation is the level of the settled formed components in the image after the initial period of accelerated sedimentation.
• Hematocrit measurement used for the ESR calculation, can be performed using at least one method such as but not limited to those described herein. It can be performed in the same system as the one with the centrifuge, or optionally, it can be performed using a physically separate instrument.
• the sample may be centrifuged to complete the sedimentation and pack the formed components into a pellet.
• the effective gravitational force on a vessel can completely cause the precipitate ("pellet") to gather on the bottom of the tube.
• the supernatant liquid is then either be decanted from the tube without disturbing the precipitate, or withdrawn with by a pipette.
• Figure 1 8B shows the logarithm of erythrocyte sedimentation rates extracted from sedimentation profiles corrected for the effect of hematocrit.
• the hematocrit correction (for example, see Figure 19A) is capable of essentially eliminating the effects of hematocrit on ESR.
• Figure 20 shows that the hematocrit-corrected and linearly-transformed Log(ESR) values obtained by the present embodiment as compared to Westergren ESR (uncorrected for hematocrit).
• the calibration has been applied (based on the fit as calculated from Fig 19), and agreement beiween the Westergren and present method is shown.
• This Figure 20 shows a demonstration of accuracy.
• Samples Fresh EDTA-anticoagulated blood samples were used. EDTA is used as this is the standard for the "Modified Westergren" method. Samples were kept at room temperature and re-suspended prior to measurement.
• Hematocrit Adjustment Samples were spun down for hematocrit packing (e.g. 5000 Relative centrifugal force (RCF) for 20 minutes), and plasma is separated from the cells. Red blood ceils were slurried with plasma from the same sample and more plasma added to give a desired hematocrit level.
• hematocrit packing e.g. 5000 Relative centrifugal force (RCF) for 20 minutes
• Westergren ESR Measurements A 1 mL sample is required to perform the Westergren ESR measurement ('Sedigren' brand tubes used, following protocol enclosed therein). Red blood cell sedimentation was observed and measured by video recording.
• a kymograph obtained using the centrifuge based methods as described herein also shows that in addition to the air/plasma interface 130 and erythrocyte/plasma interface 132, there is also a "shadow" showing a white blood cell and plasma interface 141.
• a white blood cell and plasma interface 141 shows both the red cell front red cell and a second sedimentation front corresponding to white cells.
• some embodiments of the centrifugal method may be used to sequentially or simultaneously measure white blood cell sedimentation rate which may be useful in characterizing certain aspects of patient health.
• white blood cells change their physical characteristics when they are activated and/or aggregated. Both phenomena are of great interest in e valuating white ceil function.
• White cells sediment under centrifugal force, but they sediment at a rate more slowly than red blood cells.
• the rate of white blood cell sedimentation is a function of at least one of the following: white blood cell density, shape, and aggregation state. Measuring sedimentation rate can lead to detection of one or more these changes which may then be used characterize certain aspects of patient health.
• FIG. 21 shows data indicating that the white blood cell interface is detectable due to refractive index or light scattering change rather than absorbance change.
• RBC interface position is based on absorbance change due to the hemoglobin absorbing heavily in the green portion of the wavelength spectrum. RBC interface could perhaps be similarly monitored if light of the right wavelength were used (very long wavelength).
• using light scattering or change in refractive index can also be used alone or in combination with absorbance as an alternative way of measuring interface position or for detecting some interface(s) such as for white blood cells or platelets that are not readily visible by absorbance detection alone.
• the processes described herein may be performed using automated techniques.
• the automated processing may be used in an integrated, automated system. In some embodiments, this may be in a single instrument having a plurality of functional components therein and surrounded by a common housing.
• the processing techniques and methods for sedimentation measure can be pre-set.
• that may be based on protocols or procedures that may be dynamically changed as desired in the manner described in U.S. patent applications Ser. Nos. 13/355,458 and 13/244,947, both fully incorporated herein by reference for all purposes.
• an integrated instrument 500 may be provided with a programmable processor 502. which can be used to control a plurality of components of the instrument.
• the processor 502. may control a single or multiple pipette system 504 that is movable X-Y and Z directions as indicated by arrows 506 and 508.
• the same or different processor may also control other components 512, 514, or 516 in the instrument .
• tone of the components 512, 514, or 516 comprises a centrifuge.
• control by the processor 502 may allow the a sample handling system such as but not limited to pipette sy stem 504 to acquire sample from cartridge 510 and move the sample to one of the components 512, 514, or 516.
• the sample is blood.
• Such movement may involve dispensing the sample into a removable vessel in the cartridge 510 and then transporting the removable vessel to one of the components 512, 514, or 516.
• sample is dispensed directly into a container already mounted on one of the components 512, 514, or 516.
• this may occur without having to transfer the sample into an intermediate vessel prior to dispensing the sample into a container at one of the components.
• some embodiments may use the container used for sample eollection from the subject and process the sample while it remains in the collection vessel.
• a componeni such as but not limited to a centrifuge or other sample separator, without having to further transfer the sample fluid to yet another container that is used in the centrifuge device.
• the container in which the sample was collected from the subject may have a shape conducive to enabling eentnfugaiion of small volume blood samples.
• this collection container may be sufficiently transparent on portions of at least one surface to allow for imaging of the sample therein without having to remove the sample from the collection container.
• this colleciion container may be sufficiently transparent aligned portions on at least two surfaces of the container to allow for imaging of the sample therein without having to remove the sample from the collection container.
• the ESR method is performed on the collection container, there is no loss of sample associated with aliquoting a portion of the sample to a separate container for ESR measurement.
• a method used for processing the sample may involve removing the collection vessel from a sample collection device, optionally transporting the collection vessel to a sample processing unit, and optionally inserting the collection vessel into a cartridge or directly into one of the components.
• a method for processing the sample may involve removing the collection vessel from a sample collection device, and then transporting the collection vessel to a sample processing unit.
• a method for processing the sample may involve removing the collection vessel from a sample collection device, then transporting the collection vessel to a sample processing unit, and then inserting the collection vessel into a cartridge or directly into one of the components.
• sample processing units can be, but are not limited to, those described in LIS patent applications 13/769,820 and 61/852,489, both fully incorporated herein by reference for all purposes.
• one of these components 512, 514, or 516 may be a centrifuge with an imaging configuration as shown in Figure 3.
• Other components 512, 514, or 516 perform other analysis, assay, or detection functions.
• a sample vessel in a centrifuge such as one of these components 512, 514, or 516 can be moved by one or more manipulators from one of the components 512, 514, or 516 to another of the components 512, 514, or 516 (or optionally another location or device) for further processing of the sample and/or the sample vessel.
• Some may use the pipette system 504 to engage the sample vessel to move it from the components 512, 514, or 516 to another location in the system.
• a small footprint floor mounted system may occupy a floor area of about 4nr or less, in one example, a small footprint floor mounted system may occupy a floor area of about 3m 2 or less. In one example, a small footprint floor mounted system may occupy a floor area of about 2m 2 or less. In one example, a small footprint floor mounted system may occupy a floor area of about lm 2 or less. In some embodiments, the instrument footprint may be less than or equal to about 4 m 2 , 3 m 2 , 2.5 m 2 , 2.
• Some embodiments may optionally combine the hematocrit correction techniques described herein with measurement techniques as described in U.S. Patent 6,204,066 fully incorporated herein by reference for all purposes. Some embodiments herein may pre-process the blood sample to pre-set the hematocrit value in the blood sample to a pre-determined value so that the variable due to hematocrit is removed. Some embodiments may also use traditional techniques for adjusting for hematocrit levels. It should also be understood that although the present embodiments are described in the context of blood samples, the techniques herein may also be configured to be applied to other samples (biological or otherwise).
• At least one embodiment may use a variable speed centrifuge.
• the speed of the centrifuge could be varied to keep the compaction curve linear with time (until fully compacted), and the ESR data extracted from the speed profile of the centrifuge rather than the sedimentation rate curve.
• one or more processors can be used to feedback control the centrifuge to have a linear compaction curve while speed profile of the centrifuge is also recorded.
• the sedimentation rate data is calculated based centrifuge speed. In one non-limiting example, a higher centrifuge speed is used to keep a linear curve as the compaction nears completion.
• the volume of blood used for sedimentation testing may be 1 mL or less, 500 ,uL or less, 300 L or less, 250 ⁇ _ ⁇ or less, 2.00 ⁇ ,, or less, 170 ⁇ , or less, 150 ⁇ , or less, 125 ⁇ . or less, 100 ⁇ , or less, 75 ⁇ , or less, 50 ⁇ , or less, 25 ⁇ , or less, 20 ⁇ , or less, 15 ⁇ or less, 10 ⁇ , or less, 5 ⁇ _.
• the sample collected herein comprises capillary blood.
• the sample is collected from a fingerstick.
• the sample is collected from skin at an alternate site such as a forearm, leg, ear lobe, or other location on a subject.
• some embodiments may use a collection process wherein the target area is warmed and/or wherein an initial blood sample is (or is not) wiped away (and at least a portion of the remainder is collected for processing).
• the collection of capillary blood may occur by way of a capillary tube, a tube with a syringe, or a capillary tube coupled to a collection container.
• the sample collected herein comprises primarily capillary blood with negligible amounts of interstitial fluid.
• some embodiments may use an integrated collection device with multiple collection tubes or chambers, wherein only a subset of the tubes or chambers are imaged for ESR.
• some embodiments may use an integrated collection device with multiple collection tubes or chambers, wherein only one of the tubes or chambers are imaged for ESR.
• concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
• a size range of about 1 mn to about 200 nm should be interpreted to include not only the explicitly recited limits of about 1 nm and about 200 nm, but also to include individual sizes such as 2. nm, 3 nm, 4 nm, and sub-ranges such as 10 nm to 50 nm, 20 nm to 100 mn, etc....
• a method comprising: using an accelerated blood component separation technique on a blood sample for a period of time to separate formed blood components from plasma; establishing a time -related compaction curve for at least one formed blood component in said blood sample after accelerated blood component separation has begun, said compaction curve having an initial approximately linear portion; determining sedimentation rate of the formed blood component based on at least the following: the compaction curve and a hematocrit correction factor.
• a method comprising: centrifuging a blood sample in a vessel for a period of time; establishing a time-related compaction curve for at least one formed blood component in said blood sample after centriiuging has begun, said compaction curve having an initial approximately linear portion; correcting for hematocrit effect on sedimentation rate of the formed blood component by using a hematocrit correction factor on the approximately linear portion of said compaction curve.
• a method comprising: centriiuging a blood sample in a vessel for a period of time; establishing a time-related compaction curve for at least one formed bl ood component in said blood sample after centrifuging has begun; correcting for impact of hematocrit on sedimentation rate of the formed blood component using a hematocrit correction factor based on the formula:
• Uuncorr and Uc ⁇ r are the uncorrected (raw) and corrected sedimentation rates
• ⁇ is the volume fraction of cells (hematocrit)
• e and y are empirical parameters obtained by curve fitting
• curve fitting for the hematocrit correction factor comprises calibrating sedimentation rates from centrifuge based technique with sedimentation rates from a reference technique.
• Aspect 5 The method of any one of the foregoing aspects, wherein the reference technique is the Westergren technique.
• Aspect 6 The method of any one of the foregoing aspects, wherein fibrinogen levels as high as 15mg/ml does not impact sedimentation rate measurement.
• Aspect 7 The method of any one of the foregoing aspects, wherein said blood sample is about 100 uL or less.
• Aspect 8 The method of any one of the foregoing aspects, wherein said blood sample is about 50 uL or less.
• Aspect 9 The method of any one of the foregoing aspects, wherein said blood sample is about 25 uL or less.
• Aspect 10 The method of any one of the foregoing 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 1 1. The method of any one of the foregoing aspects, wherein centrifugation uses a centrifuge configured to allow the blood sample to be visually observed during centrifugation to establish interface positions of one or more formed blood components in the blood sample.
• Aspect 12 The method of any one of the foregoing aspects, wherein
• centrifugation uses a centrifuge having a window thereon to enable visual observation of the blood sample to establish erythrocyte/plasma interface positions over time.
• Aspect 13 The method of any one of the foregoing aspects, wherein centrifugation uses a centrifuge, a light source, and an image capture device to enable visual observation of the blood sample to establish formed blood component/plasma interface positions over time.
• Aspect 14 The method of any one of the foregoing aspects, wherein compaction curve data is collected by capturing a plurality of images of interface positions of one or more formed blood components in the centrifuge vessel over the time period.
• Aspect 15 The method of aspect 14, wherem pixel positions in the plurality of images are used to accurately determine 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 capturing of images begins when the centrifuge has begins rotation.
• Aspect 18 The method of any one of the foregoing aspects, wherein compaction curve data is collected while the sample is being centrifuged.
• Aspect 19 The method of any one of the foregoing aspects, wherein centrifugation is used to obtain accurate values for the hematocrit and to correct for hematocrit impact on sedimentation rate measurement.
• Aspect 20 The method of any one of the foregoing aspects, wherein correcting for hematocrit comprises calculating a mathematical function for a plurality of formed blood component interface positions occurring in said curve, said function being operative to correct for sedimentation rate variations due to hematocrit.
• Aspect 21 The method of any one of the foregoing aspects, wherein hematocrit correction factor is determined without using data from a non-linear portion of the compaction curve.
• Aspect 22 The method of any one of the foregoing aspects, wherein hematocrit level in the sample is derived from a technique separate from centrifugation.
• Aspect 23 The method of any one of the foregoing aspects, wherem ( max and ⁇ are for fit optimization and do not relate directly to physical parameters.
• Aspect 24 The method of any one of the foregoing aspects, further comprising image transformation for conversion of a curved interface to a flat interface.
• Aspect 25 The method of any one of the foregoing aspects, wherein hematocrit correction is capable of essentially eliminating the effects of hematocrit on formed blood component sedimentation rate.
• Aspect 26 The method of any one of the foregoing aspects, wherein image transformation parameters are selected, video of formed blood component interface position is put through image transformation, and then a region of interest is chosen that covers both the whole range of positions for both air/plasma interface and erythrocyte interface.
• Aspect 27 The method of any one of the foregoing aspects, wherein for each timepoixit in the video, pixel intensity values for each row across the sample vessel within the region of interest are averaged to produce a single column representing the intensity radi lly down the sample vessel.
• Aspect 28 The method of any one of the foregoing aspects, wherein columns for each timepoint are then assembled into a kymograph.
• Aspect 29 The method of aspect 28 wherein positions of the two local maxima of the image, one representing the air/plasma interface and other the plasma/erythrocyte interface are determined.
• Aspect 30 The method of aspect 28 comprising converting pixel positions into volume occupied by the whole sample and volume occupied by red blood cells, wherein the y- position of the top and bottom of the centrifuge vessel are used as reference locations together with knowledge of the shape of the centrifuge vessel.
• Aspect 31 The method of any one of the foregoing aspects comprising converting plasma/erythrocyte interface position to the volume fraction occupied by red blood ceils and plotted against time as a centrifuge sedimentation curve.
• Aspect 32 The method of any one of the foregoing aspects, wherein a linear region of a sedimentation profile is used to extract a sedimentation rate.
• Aspect 33 The method of any one of the foregoing aspects, further comprising deriving an estimate of the sedimentation rate linearly related to the Westergren ESR, the centrifuge-derived, hematocrit corrected data further corrected using the formula: Estimated Westergren ESR 10 ( ⁇ ⁇ . ⁇ ⁇ T corrected ESR)-LOG(a))/b)).
• Aspect 34 The method of any one of the foregoing aspects, further comprising hematocrit-correcting and linearly-transforming Log(ESR) values to establish a linear graph of sedimentation rate.
• Aspect 35 The method of any one of the foregoing aspects wherein the blood sample is whole blood.
• Aspect 36 The method of any one of the foregoing aspects wherein the blood sample is an ants-coagulated sample,
• Aspect 37 The method of any one of the foregoing aspects wherein the formed blood component is white blood cells.
• Aspect 38 The method of any one of the foregoing aspects wherein the formed blood component is platelets.
• Aspect 39 The method of any one of the foregoing aspects, further comprising determining white cell sedimentation rate after centrifugation has begun, wherein measuring white cell sedimentation rate characterizes at least one of the following regarding the white blood cells: cell density, shape, and aggregation state.
• a method comprising: collecting a plurality of images of formed blood component and plasma interface positions over time from an accelerated blood sample compaction process; performing image transformation on said plurality of images to transform images with curved interfaces into corrected images with straight line interfaces; establishing a time-related compaction curve based on mterface positions in said corrected images, for at least one formed blood component in said blood sample.
• a method comprising: centrifuging a blood sample in a vessel for a period of time; collecting a plurality of images of formed blood component and plasma interface positions over time: performing image transformation on said images to transform images with curved interfaces into corrected images with straight line interfaces; establishing a time-related compaction curve based on interface positions in said corrected images, for at least one formed blood component in said blood sample after centrifuging has begun.
• a method comprising: using a programmable processor-controlled system to transfer at least a portion of a blood sample from a blood sample location into a centrifugation vessel; using a sample handling system under programmable processor control to transfer said vessel from a first addressable position to a centrifuge with a second addressable position; centrifuging the blood sample in the vessel for a period of time; collecting a plurality of images of formed blood component and plasma interface positions over time;
• Aspect 43 The method of any one of the foregoing aspects, wherein the centrifuge has a rotor with a diameter of about 15 cm or less.
• Aspect 44 The method of any one of the foregoing aspects, wherein the centrifuge has a rotor with a diameter of about 10 cm or less.
• Aspect 45 The method of any one of the foregoing aspects, wherein the centrifuge has a rotor when in motion circumscribes an area with a longest dimension of about 15 cm or less.
• Aspect 46 The method of any one of the foregoing aspects, wherein the centrifuge has a rotor when in motion circumscribes an area with a longest dimension of about 10 cm or less.
• a method comprising: centrifuging a blood sample in a vessel for a period of time; varying centrifuging speed to establishing a linear compaction curve of at least one formed blood component over the period of time until compacting has completed;
• a method comprising: centrifuging a blood sample in a vessel for a period of time; collecting at least a first single image of formed blood component and plasma interface positions at an initial time; collecting at least a second single image of formed blood component and plasma interface positions at a second time while rate of sedimentation is still linear; calculating sedimentation rate for at least one formed blood component in said blood sample based on linear sedimentation rate calculated and a hematocrit correction factor.
• a device for use with a sample comprising:
• a centrifuge having a centrifuge vessel holder configured to allow for detection of blood component interface position in the vessel holder during centrifugation.
• Aspect 50 The device of aspect 49, wherein the centrifuge has window to allow for visual observation of the centrifuge vessel holder during centrifugation.
• Aspect 51 The device of aspect 49, wherein the centrifuge an illumination source to allow for detection of blood component interface position in the sample.
• a system comprising: a centrifuge having a centrifuge vessel holder configured to allow for detection of blood component interface position in the vessel holder 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 interface position during a least a portion of centrifugation.

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• 2015
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