Patent Description:
Blood samples are often analyzed or processed by centrifuging the blood sample to separate out particular components of the blood sample into component layers. Typically, the centrifuged blood sample comprises three component layers, the top plasma layer, the bottom red blood cell layer and the middle "buffy coat" layer containing white blood cells. Centrifugation allows particular components of interest to be extracted from the blood sample by removal of the appropriate component layer.

The component layers of the blood sample are typically extracted manually in turn by a pipette. The component layer containing the component of interest is retained for analysis and the other component layers may be retained or disposed of as desired. Manual extraction of the blood sample component layers in this manner is time consuming and expensive. It also requires considerable skill as, to the naked eye, the boundaries between blood sample component layers can be difficult to distinguish. These problems are exacerbated if the buffy coat layer is the fraction of interest, as the buffy coat layer is typically relatively thin compared to the other blood sample component layers. <CIT> describes a method of determining a characteristic of a clinical sample by transmitting a beam of radiation through it. <CIT> discloses a method of determining boundaries between factions in a sample. <CIT> discloses a model based method for inspecting a sample for the presence of an interferent. <CIT> describes a method and apparatus for capturing an image of a boundary layer in a sample.

Proper illumination of the blood sample and the component layers within is critical to providing a high-contrast image which may be reliably analyzed. Commonly, reflections interfere with the image and require complex hardware or software filtering.

It is therefore an object of the present invention to provide a method and apparatus for the automated illumination and imaging of a centrifuged blood sample.

The present invention provides a blood sample processor for imaging a centrifuged blood sample and a method for analyzing a centrifuged blood sample as set out in the claims.

The pipette with a liquid level height sensor is provided for determining the actual location of the top of the centrifuged blood sample and for removing component layers therefrom.

The processor is provided to determine the actual locations of component layers of the centrifuged blood sample.

Accordingly, accurate detection of the location of the buffy coat layer allows automated collection of any of the discrete component layers of a centrifuged blood sample. Detection of the buffy coat layer may be done by optical imaging, using a camera to image the container with the centrifuged blood sample therein and algorithms to post-process the centrifuged blood sample image to quantitatively analyze the vertical location of each component layer within the container.

The features and advantages of the invention are apparent from the following description taken in conjunction with the accompanying drawings in which:.

After centrifugation, there can be distinguished a layer of clear fluid (the plasma), a layer of red fluid containing most o'f the red blood cells, and a thin layer in between. The buffy coat layer is the fraction of an anticoagulated blood sample that contains most of the white blood cells and platelets following density gradient centrifugation of the blood.

Referring to <FIG>, a blood sample after centrifugation is shown in test tube <NUM>. The blood sample has three component layers; a plasma layer <NUM> at the top of the test tube, a red blood cell layer <NUM> at the bottom of the test tube and an intermediate or buffy coat layer <NUM>. In some instances, a clean buffy coat layer is approximately <NUM> thick. Above the clean buffy coat layer is a transition buffy coat layer that is approximately <NUM>-<NUM> thick. Together the clean buffy coat layer and the transition buffy coat layer make up the buffy coat layer.

Typically, only the clean buffy coat layer is desired for processing. Accordingly, first the plasma layer <NUM> is removed. Then, the transition layer of the buffy coat layer <NUM> is eliminated. The clean buffy coat layer is then topmost in test tube <NUM> and available for removal.

To properly remove each of the layers, the layers must be adequately illuminated and distinguished. This may be accomplished by illuminating test tube <NUM> with a light source and then obtaining an image of the test tube and its contents. An illumination source or a light source is used to illuminate the contents of the test tube <NUM>. In some instances, the light source can be a monochromatic light source, while in other instances the light source can comprise multiple monochromatic light sources that are spectrally mixed. Examples of monochromatic light sources include LEDs that emit light having a wavelength of less than <NUM>. These light sources can be colored light that is produced by one or more LEDs. In further embodiments, the light source or multiple light sources can be filtered or un-filtered light.

<FIG> shows a blood sample processor providing an illumination source <NUM> in accordance with an embodiment of the invention. Tuning to obtain optimum contrast between sample layers in test tube <NUM> is accomplished by choosing the proper wavelengths of light for illumination source <NUM>. When illuminated, the plasma layer of a sample typically reflects or casts a yellow or amber tinted light having a wavelength of approximately <NUM> or greater, while the red blood layer reflects a red light having a wavelength of approximately <NUM> or greater. Therefore in order to minimize reflection and backscatter from the plasma and red blood layers of the sample, and further isolate the buffy coat layer, it is advantageous to illuminate the sample with a light source having a wavelength less than <NUM>. In some instances this light source can be a blue light with a wavelength of <NUM>, in others it can be a green light, while in still others an ultra-violet light source can be used. In each instance, however, the light source has a wavelength less than <NUM>, which maximizes reflection from the buffy coat layer and more specifically from the clean buffy coat layer.

In some instances, the illumination or light source <NUM> can have a LED or other light emitting element that emits light having a wavelength less than <NUM>. In other instances, the light source <NUM> can have multiple light emitting elements that emit light having different wavelengths. These multi-chromatic light sources can, in many instances, maximize reflection of light by the buffy coat layer by enhancing the differentiation between the buffy coat layer and the plasma and red blood cell layers, thereby increasing the resolution of the image of the buffy coat layer. For example, multiple LEDs having different wavelengths can be used to achieve light having different hues or color temperatures. Mixing light source wavelengths can also enhance the differentiation between the transition buffy coat layer and the clean buffy coat layer. In particular, certain light wavelengths increase the amount of reflection by the transition buffy coat layer, but not the clean buffy coat layer; while other light wavelengths increase the amount of reflection by the clean buffy coat layer but not the transition buffy coat layer. Mixing this group of two or more disparate light sources with two or more different wavelengths can increase reflection of both the transition buffy coat layer and the clean buffy coat layer. These disparate light sources can be different colored light sources having different color temperatures or hues.

Illumination source <NUM> is preferably high intensity LEDs, for example, Luxeon Rebel Color LEDS and, more particularly, the multi-LED blue (<NUM>) <NUM> LED boards. In other instances, other LEDs, LED packages, or light sources can be used. LEDs can be individual LEDs incorporated onto a single circuit board or can be multiple LED chips integrated into a single chip. While LEDs are preferably used, other methods could include an illumination source other than a LED, where the non-LED source is passed through one or more spectral filters to isolate light having a wavelength less than <NUM>.

Illumination source <NUM> can be positioned approximately <NUM> degrees off of the center axis of test tube <NUM>. It may be appreciated that any number of offset angles (i.e., an oblique angle to the test tube) would be appropriate for the illumination source so long as the angle is sufficient to prevent reflection and other interference. For example, in some instances the illumination source <NUM> can be positioned at any angle between <NUM> and <NUM> degrees off of the center axis of test tube <NUM>. In still other examples, the illumination source <NUM> can be positioned at any angle between <NUM> and <NUM> degrees off of the center axis of test tube <NUM>.

A digital camera <NUM> is positioned opposite test tube <NUM> for imaging of the test tube <NUM> and the blood sample within. Preferably, a color camera, rather than a black and white camera, is used because a color camera may accentuate buffy coat layer <NUM>. In some instances, a suitable camera for this purpose may be the Cognex Advantage 100Series, Part No. ADV102C from Cognex Corporation (Natick, MA). In other instances, any high resolution camera can be used. It will be appreciated that digital camera <NUM> must be calibrated prior to imaging. It may also be appreciated that illumination source <NUM> may be triggered by digital camera <NUM>.

Advantageously, almost all test tubes with blood samples for imaging will have a label. Accordingly, imaging of the blood sample may be done through the clear side of test tube <NUM> and the test tube label may be used as a reflector. Exposure time for digital camera <NUM> may be optimized based on saturation level of the camera. It may be appreciated that the exposure for digital camera <NUM> should be set below the saturation point to insure a quality image.

A light shield <NUM> may be also be provided for use in imaging test tube <NUM> with the blood sample within. The use of a light shield with, for example, a black background may reduce ambient light thereby improving imaging of the blood sample.

In order to determine the position of the component layers, test tube <NUM> with the centrifuged blood sample therein is positioned vertically opposite digital camera <NUM>. An image of the test tube <NUM> and the blood sample within is then captured by digital camera <NUM>. The image may then be processed by a suitable processor <NUM> using photo processing algorithms using pixel count to determine the relative locations of the component layers within test tube <NUM>. A suitable hardware/software system for this application may be a <NUM>-D vision system available from Cognex Corporation (Natick, MA).

Light shield <NUM> may include an external reference against which test tube <NUM> and the blood sample within may be imaged. This will provide for direct location of the layers from the image. The external reference may also be incorporated into the blood sample processor or the rack that the test tube is in.

Processor <NUM> of the blood sample processor may also control an automatic pipette <NUM>. The automatic pipette has a liquid level height sensor <NUM> for detecting the fluid surface of the blood sample. Once the actual location of the fluid surface of the blood sample is known, the relative locations may be used to set the actual locations of each component layer of the blood sample. As the dimensions of test tube <NUM> are known, the pipette <NUM> may be inserted into the blood sample to a desired location and a volume of liquid aspirated equal to the calculated volume of a particular layer. Typically, the layers are aspirated in turn starting with the top or plasma layer.

As an example, if it is desired to extract the buffy coat layer for analysis, the pipette may be used to aspirate the plasma layer <NUM> to a level just above the upper boundary of buffy coat layer <NUM>. The pipette <NUM> may then be used to aspirate the buffy coat layer <NUM> to a level just below the lower boundary of the buffy coat layer <NUM>. The buffy coat layer <NUM> of the blood sample in the pipette <NUM> may then be transferred to another container for analysis.

In a case where a sample of the clean buffy coat layer is required for analysis, the plasma layer <NUM> may be aspirated, and then the transition layer of buffy coat layer <NUM> may be aspirated. Finally, the clean buffy coat layer may be aspirated and transferred to another container for analysis.

It may be appreciated that aspiration locations for the buffy coat layer will depend on a particular application. Assays that use downstream secondary processing can afford to collect above and below the buffy coat layer to obtain the entirety of the layer. Biobank harvesting applications may "guardband" the buffy coat layer by going past the top plasma layer and stopping short of the bottom red blood cell layer to ensure minimal contamination.

<FIG> shows how the various components of the blood sample processor may be logically connected. The sample processor <NUM> is connected directly to illumination source <NUM>, digital camera <NUM>, pipette <NUM> and liquid height level sensor <NUM>. The processor <NUM> may also be provided with a user interface <NUM>. The processor <NUM> may also control the illumination of test tube <NUM> with the blood sample during imaging. In so doing, illumination source <NUM> may be either strobed or constant on. In a preferred embodiment, the sample processor <NUM> and the user interface <NUM> may be provided by a computer system. In some instances, the illumination source <NUM>, camera <NUM>, pipette <NUM>, liquid height level sensor <NUM> and processor <NUM> may be included in a single system or machine that can be used to analyze blood samples. This single system can further include a user interface <NUM> that can be used to control the single machine. In some cases, the steps of the method can be automated by a program executed by the processor <NUM> of the machine.

Claim 1:
A blood sample processor for imaging a centrifuged blood sample, comprising:
a transparent container (<NUM>) with the centrifuged blood sample therein;
an illumination source (<NUM>) for illuminating the centrifuged blood sample and positioned to illuminate the centrifuged blood sample at an oblique angle to the transparent container;
a digital camera (<NUM>) disposed opposite the transparent container (<NUM>)for imaging the centrifuged blood sample; and
a processor (<NUM>) for processing the centrifuged blood sample image and determining relative locations of component layers of the centrifuged blood sample;
characterised in that the blood sample processor comprises a pipette (<NUM>) with a liquid level height sensor (<NUM>) for determining an actual location of a surface at the top of the centrifuged blood sample.