JP2011097936A - Operator independent programmable sample preparation and analysis system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protocol and apparatus for enriching circulating tumor cells and other rare cells from blood, including debris and other components, from samples with high precision and at high throughput rates. <P>SOLUTION: This invention discloses an improved processing system from previously described semi-automated sample processing. The system is configured for sample processing of biological specimens to provide an enriched fraction suitable for detection, enumeration, and identification of target cells. The presence and quantities of such target cells in a sample specimen can utilized for screening and detection in disease such as cancer, assessing early stage pre-metastatic cancer, monitoring for disease remission in response to therapy and selection of more effective dose regimens or alternative therapies in case of relapse. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

Inventor
Teresa Bendele, Thomas Harbart, Dave Howard, Michael Kagan, Douglas Keene, Dave Lapeus, Jared Mayes, Douglas Paynter, Jerry Prohaska, Herman Rutner

Priority This application claims the benefit of US Provisional Application 60 / 503,754, filed September 18, 2003.

  The present invention relates generally to the field of automated sample processing, and more specifically, from body fluids by automated devices for further analysis because it relates to the diagnosis, monitoring and management of specific diseases, particularly cancer. Of rare cells.

  The main method and apparatus of the present invention originates from the prior art for evaluating the detection and counting of cells in a biological sample. These biological samples include, for example, blood, lymph, or cerebrospinal fluid. The biological sample can also include a buffer with spiked and cultured cells used as a control.

  Manual methods for the isolation and quantification of rare cells of peripheral blood and other biological matrices are published and well known in the art. However, automated processing is required to create a sensitive and reproducible high-throughput test protocol suitable for clinical diagnosis.

  For highly complex inspection methods, the root cause of incorrect results is the planned or random pre-analysis error, that is, the accumulation of errors that occur during sample preparation or in the pre-processing stage rather than the analysis method itself. It is well known that it can be traced back to Pre-analysis errors can appear as changes due to technology-sensitive process steps as well as normal random or systematic changes between operators. Sample preparation, particularly cell enrichment, is an example of a pre-analytical process that shows highly variable assay results when performed inconsistently. Thus, such pre-analysis automation will minimize variability and result in more consistent analysis results.

  Cell enrichment techniques for the isolation of rare cells are varied and include various manual techniques for selectively isolating target cells for analysis. Centrifugation with or without a density gradient is a common method used. However, centrifugation is not easily linked to automation without the addition of complex robotic handling of sample tubes, computer controlled centrifuges, and the like. Often, manual washing steps, including centrifugation steps, follow these procedures. If all of these manual procedures are directly automated, the end result will be a highly complex and expensive system that mimics manual processing performed by a skilled operator.

  Magnetic separation directly from an analyte of an immunomagnetically labeled target entity is an ideal mode, but is often inefficient if the target entity is a minor component in a complex mixture. Pre-preparation by magnetic labeling and collection followed by centrifugation or gradient separation may be necessary in such cases. Again, these approaches do not lead to easy automation without the addition of complex robotic handling systems for sample tubes, computer controlled centrifuges, and the like. Also, manual washing steps, including centrifugation, often follow these procedures. Again, direct automation of these manual procedures will result in a highly complex and expensive robotic system that mimics manual processing where every processing step increases the risk of cell loss.

  Several companies offer semi-automated systems that are specifically adapted to cell sorting or selection with application in cancer diagnostics and therapy. Miltenyi Biotech, Germany is a semi-automated system for collecting magnetically labeled target cells that are then further sorted or fractionated into various cell populations, including putative target cells, on an autoMACS system, flow cytometer We are developing a desktop magnetic cell sorter. However, AutoMACS requires an enriched fraction prepared by manual pre-preparation of complex analytes, such as blood, to remove most of the non-target cells. Flow cytometry also detects particulate events of a specific size, not cells, that is, without morphological confirmation of the identity of the events detected as target cells . US Pat. No. 6,046,585 issued to Quantum Design, San Diego discloses a method and apparatus for sensing and measuring small amounts of magnetic particles that bind to target entities including cells; however, these entities are rare Unthinkable.

  The semi-automated method and apparatus described above (US patent application 10 / 081,996) is originally designed to enrich circulating cells from blood and other specimens with greater accuracy than can be achieved by the manual method described above. This primarily replaces inaccurate manual steps such as quantitative fluid transfer by vortex / mixing and magnetic enrichment and magnetic washing, thus eliminating the steps typically involved in centrifugal density gradient separation. Achieved. The suction process, usually performed manually, has also been automated. The procedure required an operator to intervene regularly throughout the process, thus causing variations in process timing. While the system has general applications for sample processing of various materials, especially biological specimens in research settings, a more automated system that incorporates a system of checks to prevent reagent or sample loss greatly improves preparation However, it will increase the integrity of the sample for clinical use.

  The disclosed device is a substantial improvement over the semi-automatic detection of 10 / 081,996. In this application, operator intervention during processing is eliminated, thus reducing sample preparation time, which reduces associated errors, and is unified by more precise control of individual processing steps. The improved system provides an enriched fraction suitable for target cell detection, enumeration and identification by various analytical methodologies. The presence and amount of such target cells in a sample specimen can be utilized for screening and detection in multiple types of diseases that are not limited to metastatic cancers and cardiovascular diseases. In addition, the isolation and analysis of these target cells can be used to identify multiple diseases such as, for example, cancers with early stage pre-metastatic cancer, monitoring response to treatment and selecting more effective usage or alternative therapies. It is important for diagnosis when evaluating the above-mentioned aspect.

  The present invention provides devices and methods for protocols that serve as a means for enriching circulating cells, cellular components, and / or target entities from blood and other analytes, which are more rapid, Fully automated and independent of manual operation in previous semi-automatic analysis methods. Thus, the present invention eliminates most of the errors due to the user and reduces assay variability. The present invention discloses a novel automated sample processing system for preparing samples for analysis that improves upon prior applications (US patent application 10 / 081,996). This is achieved in part by computer controlled fluid control, motion control, error detection, and retrieval.

  The inventive aspects of the present application are embodied in an AutoPrep system. For this system, sample processing includes two sub-steps. The first step takes place before starting the batch run and confirms that all the necessary components are present. The system evaluates the inventory of required items based on the user's request to process many samples. Through an integrated data storage device (iButton, Dallas semiconductor) and instrument sensors in a test-specific reagent carrier, the system evaluates an inventory of specific items needed to process a group of samples. The inventory includes, but is not limited to, sample tubes, system fluids, liquid waste, reagents, and sample cartridge (77) devices (US patent applications 10 / 074,900 and 10 / 303,309). During the first process, the device indicates the position of the height of the red blood cell layer in each tube of centrifuged blood and aspirates the protoplasm aspirated at a preprogrammed distance above the red blood cell layer . This process must be successfully completed in order to further process the sample.

  The second of the two processes enriches the target cells in the aspirated sample as determined by the reagent pack and associated protocol software. Enrichment is achieved by sequentially reducing the amount of fluid at each process step until a 320 uL sample volume is achieved. The sample is repeated in a series of nine separate stations. The prepared sample is subjected to a series of steps resulting in enriched rare cells for each sample in the group. Station 1 enforces the cell capture reagent to bind to the target identified in the reagent pack. Stations 2 and 3 perform a magnetic incubation that allows the coated magnetic particles to move through the sample in the presence of a magnetic field and bind to target cells. Station 3 initiates the separation process by magnetically pulling the captured cells to the inner wall of the sample tube. Station 4 collects the captured cells and unbound ferrofluid at a specific location within the tube, aspirates all non-magnetic components in the sample tube, and draws the magnetically labeled fraction by fluid flow. Complete magnetic separation by resuspension. Station 5 performs magnetic washing by adding a permeabilizing reagent, a dissociation reagent and an appropriate staining reagent (membrane, cell nucleus, etc.). Station 6 continues with staining and incubation. Station 7 performs further magnetic washing and includes removal of staining reagents. At station 8, the cells are sedimented in preparation for an unbound ferrofluid fraction (ie, separating unbound ferrofluid from cells bound with ferrofluid). This step is necessary to reduce interference with uncoupled magnetic fluid fluorescence measurements. Station 9 reduces the amount of unbound ferrofluid present and dispenses cell preservatives, such as those described in US patent application 10 / 780,349, in a buffer solution to enrich the enriched cells in solution. For transfer to the sample cartridge (US applications 10 / 074,900 and 10 / 303,309).

  Within the two discussed processes, motion control elements have been developed to ensure the correct and accurate location of probes, sample tubes, and reagents during work. The magnetic shuttle device allows for computer controlled application to a specified step sample in a magnetic gradient process. The tube rotation system rotates the tube to facilitate the total magnetic incubation and separation required for the process.

  The system described by the present invention has a series of sensors that confirm that the entire sample has been processed correctly. These processing steps include, but are not limited to, protoplast aspiration, magnetic separation, post-separation aspiration, reagent addition, expected intermediate sample volume, and collection transfer to the sample cartridge (77).

  Finally, the system incorporates several facilities within the computer interface, which is essential to provide automatic and rapid sample preparation. A cleaning routine and associated fluid are also included to enable unattended maintenance with minimal operator work.

  Thus, this automated rare cell enrichment processor improves process efficiency, reduces operator error, and improves the overall accuracy of the previously described manual or semi-automated process. The present invention discloses a novel automated sample system having general utility in accurate sample processing. In particular, the system embodies a centrifuge for reliable and efficient sample processing of biological specimens such as blood and is enriched with a cell enriched fraction suitable for detecting rare target cells. I will provide a. The inventory and identification of these target cells is accomplished by specific analytical methods such as, but not limited to, the imaging systems described in US6365362 and US6013188. The present invention thus embodies the use of the above device in assessing the presence and quantification of such target cells in a sample analyte. Although all circulating cell types are considered in the use of the present invention, this device is utilized for screening and detection of early stage pre-metastatic cancer, monitoring response to therapy and selecting more effective usage or alternative therapies. can do. Epithelial cells, endothelial cells, bacterial and fungal cells are also contemplated in the present invention. Furthermore, the present invention is useful in assessing the presence and quantification of such cellular components as components and circulating debris.

The flowchart of sample pre-processing. A series of steps from blood collection to presentation of a sample for batch processing is shown. The flowchart which shows the process included when setting a batch run. FIG. 4 is an illustration of an instrument reagent pack and reusable carrier. The front and top view of system arrangement. Magnetic arrangement for AutoPrep magnet. Panel A shows the field lines and contours for a 180 degree dipole magnet (stations 4, 5, 7, and 9). Panel B shows the field lines and contours for a 70 degree tripole arrangement (stations 2 and 3). Relationship between holding force and meniscus shear force for 180 dipole configurations. Continuous effects on samples at individual stations. Station 1 aspirates protoplasm and adds capture reagent. Stations 2 and 3 are magnetic incubations. Station 4 is the first magnetic separation and resuspension part. Station 5 is the addition of staining reagent. Station 6 is a staining reagent incubation. Station 7 is magnetic cleaning. Station 8 is the first step of removing unbound magnetic particles. Station 9 is the second step of unbound ferrofluid removal and final resuspension and sample transfer to the cartridge.

Detailed description of the invention:
Tumor cells are often present in the blood of cancer patients at a very low frequency (<10 cells / ml) and can provide clinically useful information. However, the difficult procedures necessary to detect and quantify the presence of circulating tumor cells lead to high levels of variability in the results. The system of the present invention assists in the processing and analysis of 4-30 ml blood samples against tumor cells and other rare cell fractions of epithelial origin.

  Target cells are labeled by magnetic means, separated from blood, and captured cells are fluorescently labeled to allow detection and differentiation from non-target cells.

  The apparatus of the present invention eliminates a substantial number of user-required steps and is a suitable analysis chamber for use in an image analysis system (eg, but not limited to the chamber described in US 10 / 074,900). To produce a 320 ul liquid sample placed in For example, in CellSpotter®, the chamber is placed in a magnetic instrument that directs magnetically labeled cells in the sample to an optically transparent plane of the chamber. The magnetic instrument and chamber are then placed on a fluorescence microscope equipped with a computer controlled filter selector and a digitally controlled X-Y-Z stage, as illustrated in the CellTracks® automated fluorescence imaging apparatus.

The present invention is, in part, an automatic diagnostic system that includes steps a through f of the following procedure, all performed by the disclosed apparatus.
a. detection of the location of protoplasmic red blood cell migration in the sample,
b. Removal of protoplasts
c. for the purpose of forming a magnetically labeled target, add magnetic particles covered with a binder that recognizes specific determinants for the target;
d. Incubate and selectively collect, thereby magnetically separating the labeled cells from unwanted non-target and matrix components and removing the non-target cells;
e. selectively staining the collected label and the remaining non-target with multiple fluorophores to allow discrimination of the target from the non-target;
f. magnetically wash the stained target,
g. fractionating bound cells from unbound ferrofluid,
h. transfer the collected labeled target to an analytical device (eg, a sample chamber for analysis), and
i. The collected cells are analyzed by appropriate analytical methods for the purpose of characterizing, identifying and enumerating the targets present in the specimen.

  The last three steps (steps g through i) may be performed in a novel sample chamber (US 10 / 074,900 and US 10,303,309) incorporated in the following patents and co-pending applications; US 5,186,827, US 5,698,271 , US 6,120,856, US 6,551,843, US 09 / 702,188, US 10 / 449,355, or on the CellTracks system (US Patents 5,985,153; 6,013,188; 6,136,182 and pending application 10 / 602,979).

  The present invention is an improvement in the methodology, apparatus and software previously described (10 / 081,996) for automated techniques-sensitive processes in cell enrichment. This improvement provides programming for further reduction of target cell loss, reduced assay variability, improved process reliability and efficient and effective performance.

Pre-batch preparation Blood sample preparation begins with sample collection (Figure 1). Individual samples are appropriately labeled using bar code labels or other means of unique sample identification.

  The basic process improvements involved in sample handling and approaches to handling specific error situations are outlined below. These improvements apply to probe priming and washing, reagent addition, post-separation aspiration and magnetic separation methods common to several stations in the automated process of the present invention. Any errors that occur during batch preparation are reported to the operator for correction before the preparation process continues. When processing samples obtained from a patient, it may be necessary to redraw the patient's blood due to individual sample loss from system failure. This situation delays the process of having the patient submit a new sample and providing the clinician with the results. Minimizing the possibility of unsuccessful samples is solved to some extent by the improved aspects of the present invention.

  If the process stops due to missing reagents or devices (ie, cartridge (77)) required for processing, the sample may be processed incompletely. For this reason, one example behind current systems is that it is possible to evaluate the inventory of these items before attempting to process the samples. For example, system fluid level, liquid waste container level, reagents, and sample cartridge (77) are inventory items that are checked to avoid downstream sample processing errors.

  Inventory valuation is accomplished through several means such as, but not limited to, data storage devices and sensor subsystems (FIG. 2). Before starting a batch run, the operator needs to attach a sample tube with either a barcode identifying the sample ID or a unique manually entered accession number. The device will move the tube to a barcode reading station and will attempt to read the barcode. The reading takes place immediately after the operator confirms that the tube has been installed and allows it to be corrected before the next tube is installed. If the bar code cannot be read or is not present, the device informs the operator and presents the tube for readjustment or relabeling or manual entry of the accession number.

  The supported barcode formats are one of the following: Code 128, Codabar, Interleaved 2 of 5, and Code 39.

  The sample cartridge (77) is barcoded and read to verify the cartridge (77) and each sample cartridge is properly installed. Based on successful reading and recording of data to individual cartridge data storage devices (Figure 1, bottom panel), the system proceeds with the batch process.

  The reagent pack data button is read and used to verify that sufficient reagent inventory in the reagent pack exists until sample processing is complete (FIG. 3). The data button (31) used in the reagent pack (32) has 16K bits of EEPROM storage and contains all reagent lots, expiration dates, and inventory information required by the associated system.

  Prior to the start of the batch run, the red blood cell layer in the sample tube is measured at station 1. An effective red blood cell layer is defined in the test definition software for specific testing. For a sample volume of 7.5 ml, 1.5 ml is equivalent to 20% hematocrit and 4.5 ml is equivalent to 60% hematocrit.

  In the preparation of protoplast aspiration, move the tube to the protoplast aspiration position of station 4. The fluid level is confirmed using a Station 4 aspiration probe and an integrated liquid level sensor. If the fluid is not in range, the operator is informed about corrective action. This acts as a check for the proper volume of the sample before the start of the process.

  The Station 4 aspiration probe aspirates to a pre-programmed distance beyond the red blood cell layer, as determined in advance, and all aspiration is performed on all samples as a batch before subsequent processing begins . Prior to initiating batch sample processing, the operator can correct errors detected by this process.

Batch Processing Once a batch pretreatment has been successfully completed for all attached samples, an individual batch run begins. The tubes are subjected to a series of 9 stations for sample processing, and up to 8 unique samples are processed sequentially into the following protocol. A general view of the system layout is shown in FIG. 4 by a front and top view. According to the prediction described in the following description, each station will be equipped with two probes; one for aspirating waste fluid and one for reagent collection and dispensing. Each station that introduces a magnetic field to the sample is equipped with a magnet assembly and shuttle mechanism to draw or pull the magnetic field away from the sample.

Station 1—Reagent Addition and Mixing At Station 1, reagents such as buffers, ferrofluids (ferrofluids), and capture facilitating reagents (ie streptavidin) are added to reagent probes (51) (US 6,623,982 and US 6,620,627). Dispense through.

  The station 1 reagent probe (51) is used to aspirate the first reagent (ie buffer). The first essential reagent bottle is properly placed in the carousel (52) to find the fluid level and aspirate the reagent. This procedure is repeated for the second and third reagent bottles (ie, ferrofluid and capture enhancing reagent, respectively). For each subsequent aspiration, separate each reagent so that a small air gap is defined in the method. A similar air gap is used in each subsequent station procedure that requires aspiration of multiple reagents. These air gaps reduce the possibility of cross-contamination of reagents during multiple collection methods.

  A reagent probe is sent to detect the surface of the sample using capacitive liquid level sensing. Once the fluid level is known, the probe tip remains below the surface of the fluid, minimizing bubble formation. The probe (51) will penetrate the initial volume of red blood cells and confirm that the reagent is dispensed appropriately for the reagent and sample mix. The probe (51) tracks the fluid level upward to minimize exposure to the sample outside the probe (51). Following dispensing, both the inside and outside of the probe (51) are washed to reduce sample carryover effects.

Station 2—Magnetic Incubation FIG. 5 (Panel B) shows the field lines and contours for the 70 degree tripole arrangement of magnets at stations 2 and 3, where the objective is to concentrate either ferrofluid or cells. It is not a collection but to maintain a sheet of particles.

  Station 2 is the first of two magnetic incubation stations. Place the tube in a magnetic field and pass the sample through the ferrofluid. When sufficient time for collection has elapsed, the magnet is pulled apart and the tube is rotated 180 degrees. The magnet is drawn again and the collection process begins again. Thus, magnetic incubation is a combination of circulation of tube rotation and magnet attraction. Incubation may include one or more cycles of tube rotation with a distant / close arrangement of magnets. A typical cycle is every 3 minutes and is programmable within the method for each application.

Station 3—Magnetic Incubation and Collection Move the tube to Station 3 to continue the magnetic incubation. The circulation is similar to that of station 2, but the magnetic field circulation is modified during the process of starting the collection of ferrofluid and captured cells on one side of the sample in the preparation of station 4. At the end of station 3, the magnetic field is pulled away from the tube and the tube is moved to station 4.

Station 4-Collection and Sorting Station 4 also includes a magnetic separation procedure that attracts the magnetic field and slowly rotates the tube. FIG. 5 (panel A) represents the field lines and contours for the 180 degree dipole magnet used in this station. The purpose of this station was to collect the captured cells and unbound ferrofluid, while pulling them both down to a lower level in the sample tube and resuspending the cells with 3 mL fluid flow. It becomes cloudy. The original sample volume is larger than the resuspension flow at this station. Thus, the cells and magnetic particles must be at a physical height consistent with the volume of the dispense volume. Since resuspension is performed by fluid flow, unlike vortexing in the manual method described previously, it is important to place the cells in a predictable and known location. As the sample tube is rotated relative to the high gradient portion of the magnet arrangement, the magnetic field gradient places the cells along the high gradient region defined by the north and south pole contacts.

  Suction after separation starts at station 4 tube when rotation stops. The fluid level in the tube is known. The peristaltic pump then slowly aspirates fluid with a minimal amount of turbulence by the probe (51) while maintaining a small constant depth during aspiration. Careful balance between magnetic retention and fluid shear forces is necessary so that rare cells are not destroyed or removed from the tube surface and aspirated and discarded (see FIG. 6). When the suction is finished, the probe (51) moves up to a position above the original sample level.

  Subsequently, the post-separation aspiration is verified by the aspiration probe (51) to find the fluid level. The fluid level should be above the bottom of the tube. When confirmed, the station 4 suction probe (51) is raised to the current storage position (home).

  A side port reagent probe (51) is prepared for wash resuspension dispensing. Using side port reagent probe (51), wash for resuspension dispense, wash dilution buffer and system buffer dispensed into capture cells resuspended along the conical wall of the tube Complete with a 3 ml mixture. The reagent probe (51) is placed above the center of the tube. After the Station 4 magnetic field is pulled away, the collected cells are resuspended by the fluid flow. After completion, move the sample to station 5.

Station 5-Cell Staining and Sample Volume Reduction Station 5 operation consists of magnetic washing and addition of ferrofluid separation reagent, permeation reagent and staining reagent. Pull the magnetic field and rotate the tube to perform magnetic separation as in station 4. Station 5 magnets are shorter than those of station 4. Thus, the magnetically labeled cells are collected at a lower location in the sample tube, thus allowing the cells to be resuspended at a lower dispense volume than that used at station 4.

  Station 5 tube rotation is stopped and the aspiration probe (51) is used to detect the fluid level in the tube that provides a check for proper dispensing of the Station 4 reagent volume (3 ml). The volume in the tube should be equivalent to the wash-dispense volume. If the fluid level is not within the predetermined range, the station 4 reagent addition fails and an error is reported.

  The magnet is attracted and the post-separation suction process is completed by the station 5 suction probe (51). As noted above, maintaining a balance between magnetic holding force and fluid shear force is necessary to prevent damage to rare cells. The peristaltic pump moves down to maintain the probe depth during aspiration as described in station 4 to aspirate fluid (typical aspiration rate is about 120 to 250 ul / sec) ). When finished, the station 5 suction probe (51) returns to the home position.

  The probe (51) is then used again to verify that aspiration has been successfully completed. For cell resuspension, the reagent probe (51) collects the reagent described above and places it above the outer quadrant of the tube, pulling off the magnetic shuttle. The reagent probe tip is placed just above the outer quadrant surface of the sample tube, which coincides with the top of the collected cells along the tube wall.

  Dispense reagents. Further mixing is achieved through a series of small suction / dispensing cycles and small tube rotation operations while the probe is below the fluid level. This improves reagent mixing and, once a separation reagent (biotin) is added, helps ferrofluid separation (U.S. 6,623,982 and U.S. 6,620,627).

Station 6-Staining Incubation Station 6 includes incubation for staining and disaggregation of magnetic particles. Here, the cells are stained.

Station 7—Magnetic Wash and Sample Volume Reduction In Station 7, the cell experiences a magnetic wash and reagent removal. The liquid level in the sample tube is detected via volume level sensing. Compare the capacity of the tube with the predicted value. If the capacity is not as expected, an error is reported to the operator. Station 7 magnets are shorter than those of station 5. Thus, the magnetically labeled cells are collected at a lower location in the sample tube and the cells are resuspended with a lower dispense volume than that used in station 5.

  1 ml of protein-based buffer is collected from the reagent pack (52) and added to the sample to dilute the reagent added at station 5. The sample is mixed in the same manner as the suction / dispensing method described for station 5.

  As with stations 4 and 5, the magnetic field is drawn and the tube is rotated to perform the magnetic separation procedure, however, the station 7 magnet is shorter than that of station 5 in the vertical direction. Device buffer and reagents are aspirated from their associated bottles.

  Prepare for aspiration after separation. The fluid level in the tube is known by the suction probe (51). If the fluid level is not within the predetermined range, station 5 and / or 7 reagent addition is unsuccessful and an error is reported.

  The post-separation is completed using station 7 suction probe (51). The syringe pump specific to this station slowly removes the fluid in the tube by moving the probe (51) downward to maintain the probe depth during aspiration. Station 7 suction probe (51) is raised and returned to the home position.

  Post-separation suction verification is completed by the suction probe (51) to determine the fluid level just above the bottom of the tube. For preparation of the resuspension dispense, the Station 7 magnet is then pulled away from the tube that is rotated 180 degrees so that the magnetic particles are farthest from the magnet. The reagent probe (51) is placed above the outer quadrant of the tube and on the magnetic particle pellet. The reagent probe (51) is then lowered to a position where the tip is just above the collected cells.

  Using the reagent probe (51), the reagent is dispensed along the conical tube of the tube to resuspend the captured cells. Further mixing is achieved through a series of small suction / dispensing cycles and small tube rotation motions, during which time probe (51) is below the fluid level. This improves reagent mixing and helps with ferrofluid separation. The tube is then moved to station 8.

Station 8-Cell sedimentation Station 8 allows cell sedimentation and ferrofluid removal. Here, the cells settle during the process in preparation for magnetic ferrofluid fractionation. After this, the tube is transferred to station 9. Depending on the relative density of the cells with respect to the surrounding buffer, the cells settle at a rate of about 6 um / sec, while unbound and separated ferrofluids are in suspension due to their colloidal nature. stay.

Station 9-Ferrofluidic Fractionation and Cell Migration In station 9, unbound ferrofluid is aspirated and discarded, and the magnetic fraction of cells is transferred to the analysis cartridge (77).

  The reagent is aspirated into the moving probe (51) by placing the necessary bottles at station 9.

  Magnetic separation for ferrofluid reduction is achieved by pulling the magnet and pulling and maintaining the settled cells on one side of the conical portion of the tube. The magnet attracts a programmed amount of time to avoid collecting unbound ferrofluid. The field lines and contours for the 180 degree magnetic configuration used in this station are shown in Figure 5 (Panel A). This is similar to the arrangement at stations 5 and 7 with smaller magnets. Like the previous station magnet, the station 9 magnet is smaller than that used in station 7, thus reducing the vertical position of the magnetically labeled cells and allowing re-suspension with about 250 uL of fluid .

  Preparation for post-separation aspiration is accomplished using the aspiration probe (51) to find the fluid level in the tube. This is a check for whether the station 7 reagent dispensing process has added the appropriate volume. The volume in the tube should be within a predetermined range. If the fluid level is not within range, the station 7 reagent addition has failed and an error is reported.

  The post-separation aspiration is completed (100 ul / sec) by the aspiration probe (51) placed at the bottom of the sample tube.

  The preparation for the dispensing of fixative (U.S. 10 / 780,349) resuspension follows. The magnet shuttle is pulled apart and the moving probe (51) is lowered to a position where the probe tip is above the center of the tube. The resuspension is dispensed via the transfer probe (51). Reagent dispensing is from the center of the tube containing the resuspended capture cells. A series of aspiration / dispensing cycles are performed to improve cell mixing and resuspension.

  For preparation for transfer to the cartridge (77), find the fluid level in the tube and check for the appropriate volume from the station 9 dispense. The volume in the tube should be reagent. If the fluid level is within range, the station 9 reagent addition has failed and an error is reported.

  The fluid is transferred to the sample cartridge (77). Using the moving probe (51), the entire contents of the tube are aspirated by placing the probe tip on the bottom of the tube during aspiration. The moving probe (51) is moved in the X direction to the vertical home position and to the position above the target cartridge (77). The moving probe (51) is lowered into the cartridge (77) and the moved solution is dispensed into the cartridge (77), during which the fluid level is upward.

  The remaining cells are washed out of the sample tube and moved. Aspirate 75 ul of system buffer into the moving probe syringe. Buffer is then dispensed to the bottom of the sample tube to wash away any remaining cells that may remain during the initial transfer. From this, the system buffer is again aspirated and transferred to the sample cartridge (77). In order to cause any bubbles in the cartridge (77) to escape, the volume in movement will overflow the cartridge (77) and any bubbles will exit through the entry port. Furthermore, when dispensing a sample into a chamber in the presence of a magnetic field supplied by a cartridge device, all magnetically labeled entities in the sample are evenly distributed across the analytical surface of the sample chamber (US 10 / 074,900 and US 10 / 303,309).

  The system software writes the data for each sample to the cartridge data button. This data includes a flag indicating that the data is now valid and is a time stamp when processing the sample.

Motion Control Overview Operates at each appropriate station to ensure that no probe containing reagent passes over the opening of the other bottle, tube, or cartridge (77) during operation. Control requirements have been improved. This requirement reduces potential cross-contamination of reagents or samples while the system is on the circuit board. This requirement is also met to some extent by ascertaining the position of the probe (51) in a good location during reagent, sample, or cartridge transfer operations.

  To this end, the process ring (53) moves the sample tube (54) relative to the magnet assembly (55), tube rotation drive, suction and reagent probe (51), sample tube barcode reader (56), and operator. Put on. Accurate and accurate placement in the X axis is necessary for placement of the probe tip relative to the sample tube. This should be directed during aspiration when the probe (51) should typically be in the center of the tube, and the probe (51) should be directed to the magnetically collected row of cells Applies to both resuspension reagent dispensing of time This is also a measure of the accuracy of the liquid level sensing in determining whether an appropriate amount of fluid is in the sample tube at a particular location, so vertical (Z direction) probe position accuracy and Accuracy is important at some stations.

Process Ring Sample Transfer Factors related to accuracy, accuracy and process resolution affect process ring placement. These include electromechanical drive mechanisms and position sensing systems. Proper aspiration and resuspension will allow a +/- 1.0 mm position of the sample tube relative to the probe tip. For this reason, process resolution is specified not to be more than 0.25 mm / process.

  Process ring drives are designed to move accurately and smoothly at both low and high speeds. After protoplast aspiration, the ring can be moved at high speed with an acceleration and deceleration profile that avoids sample perturbation in the tube. After completion of protoplast aspiration, a normal single position transfer during batch processing is completed in less than 2.5 seconds. All faster half-turn movements are typically completed in less than 6 seconds.

Carousel-cartridge transfer Accurate operational control is required for the carousel (57). The carousel places the cartridge (58) relative to the moving probe (51), the cartridge barcode reader, and the operator.

Probe Transport The probe (51) has a vertical motion of about 145 mm based on the height of the 15 ml sample tube.

Accurate and accurate Z-axis vertical position of the aspiration and reagent probe (51) with respect to the sample tube, reagent bottle, cartridge and wash bowl is required as follows.
a. The probe (51) vertical position evaluates the fluid level in the container while the fluid explores in the sample tube and reagent bottle.
b. During aspiration after separation, the aspiration probe (51) follows the fluid level down and stops very close to the bottom of the sample tube to aspirate the entire contents of the tube.
c. During reagent collection, the fluid level follows downward during aspiration, during which time the fluid level is detected and the probe tip is not in contact with the bottom of the reagent bottle.
d. For resuspension dispensing, place the reagent probe tip at a height above the target location on the conical tube wall and direct the dispensing flow to the row of magnetically collected cells.
e. For the final transfer to the cartridge (77), the probe tip starts near the bottom of the cartridge (77) and follows the fluid level upwards during dispensing.

  The probe (51) vertical requirements are directly related to the mechanical requirements for holding the mentioned container. For example, the process ring and tube holder need to place the tube at a certain known height for probe calibration. This places constraints on the planarity and tilt of the process ring itself.

  Similar requirements apply to reagent transfer. Each reagent probe (51) will have a calibrated bottom distance relative to the reagent bottle. If only one bottle type is used for most reagents, one bottle calibration is required for all bottles (ie 4 ml).

  An accuracy of +/- 0.127 mm is required to place the probe tip relative to the bottom of the sample tube. This ensures complete fluid removal during post-separation aspiration. For this reason, process resolution for placing the probe tip against the tube or cartridge (77) bottom is achieved in a dual probe transport model with the following designations: 1.8 degree stepper motor (200 steps / rev), 1: 1 drive rate, 12.7 mm lead screw, 12.7 mm / rev / 200 process / rev = 0.063 / process (0.032 mm / half process).

  In order to achieve a full vertical stroke of 6 seconds, a 25 mm / second vertical movement was established.

  The X axis for aspiration and reagent probe (51) controls the position of the probe tip radially relative to the tube, reagent bottle, cartridge and wash bowl. During fluid exploration and post-separation aspiration in the sample tube, the probe radial position is used to center the aspiration probe (51) in the tube. During reagent collection, the probe radial position is used to center the reagent probe (51) in the reagent bottle. During resuspension dispensing, the reagent probes (51) are placed radially to direct the dispensing flow to a row of magnetically collected cells on the conical wall of the tube. Then, with the final movement to the cartridge (77), the moving probe (51) is accurately placed and centered at the probe tip at the cartridge chamber opening. For all probe cleaning, the probe (51) should be well placed in the cleaning bowl. These probe radial placement requirements are directly related to the requirements for the mechanism holding the container. For example, the process ring must place all eight tubes constant relative to the radial position of the probe (51). Similarly, the carousel (57) needs to place all eight carriers at station 9 consistently with respect to the radial position of the moving probe (51).

  The probe transport (59) has a 150 mm x-axis stroke and the reagent probe (51) has a distance of 35 mm inside from the outer end of a 15 ml tube on the reagent pack for bottle opening access be able to.

  Similar to the above, factors that affect the probe radial position are electromechanical drive mechanisms and position sensing systems such as sensors, flags and the like. The most stringent requirement is access to the cartridge chamber opening for the moving probe tip (a 1.27 mm diameter probe tip to a 2.33 mm diameter opening). This is about 0.38 mm per probe X movement along the side. The cartridge is accurately placed in the cartridge holder. The cartridge holder plate is precisely placed on the mounting surface on the carousel. The probe tip can be repeatedly placed on the cartridge opening within about +/− 0.25 mm. For instrument calibration purposes, the process resolution is not more than +/- 0.127 mm.

Magnet shuttle The magnet shuttle places the magnet assembly in the proper position relative to the sample tube and engages the tube rotation system. For magnetic separation, the magnet contacts the sample tube during separation for maximum magnetic field penetration in the sample and maximum holding force on the tube wall during post-separation suction. In order to maximize the magnetic holding force, the gap between the magnet and the tube must be minimized by any design that has no variation due to inaccurate machine calibration. In the present invention, the custom-formed tube has a thickness detail of 0.89 mm +/- 0.127 mm.

  Spring up all magnets to ensure positive contact with the tube. The spring-up design compensates for the concentricity variation of the tube and accommodates 0.050 ”movement. This spring force is low enough not to interfere with tube rotation. The magnetic shuttle has a range of motion of 5 cm. Place the magnet far enough to index the process ring so that it does not affect the tube when pulled apart.

  Although the position of the magnet shuttle depends on the electromechanical drive mechanism and position sensing system, most of the accuracy requirements are mitigated by the spring-up of the magnet shuttle.

Tube Rotator The tube rotation mechanism is designed to rotate the tube to facilitate magnetic incubation and separation. For incubation, the tube is rotated relatively fast when the magnet is withdrawn. For separation, pull the magnet and rotate the tube very slowly, exposing all of the tube to a high gradient magnetic field along the conical wall of the tube that matches the high gradient magnetic field of the magnet assembly. Manipulate the collected cells in a row.

  With the primary purpose of rotation to locate magnetically collected cells, there are no stringent location requirements for the mechanism. At very slow speeds, a relatively slow movement needs to have a reasonably fine process resolution for any movement. For example, if rotated a large distance, 10 um cells against the tube at a high magnetic gradient point will move suddenly. Due to the small process resolution, the tube rotates by a very small amount so that the collected cells can slide or roll gently to a new high magnetic gradient position.

Fluid Design A further embodiment of the present invention is fluid design. All fluid elements are placed in a location that will mitigate the splashing, spraying and / or spilling of fluid into critical mechanical or electrical subsystems. When applicable, drip trays or shields are designed to further reduce these problems.

  System buffer is used as the backing fluid used for reagent dispensing. As reagents are aspirated into the tubes, they cover the tubes and dilute with the system fluid. By adding a small volume of backing fluid and distributing the volume, the remaining amount of the desired reagent that would otherwise remain on the surface of the tube is flushed to improve the overall recovery of specific reagents .

  Design suction design guidelines for all parts of the fluid system in the range of about 50 ul / sec to 1250 ul / sec. The aspiration line must be prepared with system buffer before aspiration to allow uniform aspiration. In order to avoid suction rate fluctuations, the pump head must contain a sufficient number of rollers. For example, Cavro Smart Peripump has 8 rollers. The suction duration is generally controlled by time or by pump head rotation. A notch is provided in the tip of the aspiration probe to prevent blocking the tip against the bottom of the sample tube. The typical inner diameter for the aspiration probe tip is 1.27 mm at the probe tip and 1.78 mm at the probe body.

  When aspirating fluid after magnetic separation, fluid shear forces are minimized as the fluid level decreases in the tube. An acceptable shear force cannot be calculated because the antibody density of the cells will not be constant. If the fluid is aspirated too slowly, the cells outside the fluid can dry out and stick to the wall of the tube, thus reducing the ability to collect them. Conversely, if suctioned too fast, cells can be lost or damaged. Therefore, these speeds must be optimized. Optimization is limited to magnetic addition of cells, magnetic permeability of ferrofluid particles, fluid meniscus velocity during aspiration washing, proximity of the probe tip to magnetically retained cells and magnetic field gradient that keeps cells in place Not dependent on them.

  The suction design details for the waste suction subsystem are set to aspirate at a nominal rate setting of about 50 ul / sec to 1250 ul / sec. Suction mark after separation, remaining amount in the tube is less than about 6 ul.

Waste aspiration Waste aspiration encompasses protoplast aspiration, most post-separation aspiration and probe washing. In most instances, these suctions are performed by a peristaltic pump through a continuous pinch valve. This type of pump and valve controls the flow of fluid but does not contact the fluid itself. This avoids degradation of the fluid elements that are sensitive to contact with biological material.

  Protoplasmic aspiration is performed in two steps after the red blood cell layer is detected by the system. First, most of the protoplasm is aspirated (approximately 450 ul / sec) using a peristaltic pump until approximately 1.5 to 2 ml of protoplasm remains. Second, the peristaltic pump slows down to aspiration at about 110 ul / sec, removing an additional 1 ml, leaving about 0.5 ml of protoplast in the tube. A slower speed is used to minimize disruption in the sample when the probe tip is near the area of the sample containing the target cells.

Probe Cleaning Probe cleaning is another aspect of fluid design and consists of drawing system fluid through a suction probe (51) or a cleaning bowl. When cleaning the probe (51) or draining the cleaning bowl, the pump operates at a fairly high speed (1 ml / sec). Techniques for cleaning the outside of the probe (51) include creating a vacuum with a peristaltic pump while the pinch valve is active (closed). When the pinch valve is turned off, fluid is quickly aspirated from the wash bowl and improves the probe cleaning of the outer surface of the probe (51) due to the high meniscus velocity in the wash bowl.

Reagent dispensing Add reagent to the sample tube at stations 1, 4, 5, 7 and 9. Some of these additions are for resuspension using 150 ul to 3 ml.

  The reagent probe tip for dispensing is designed to achieve the appropriate flow rate. Typically, the inner diameter at the tip is about 0.9 mm to 1.27 mm depending on the probe design. Variations in the inner diameter of the dispensing probe tip will dramatically change the fluid velocity exiting the probe tip.

  The reagent dispense at station 1 is a large volume dispense of 6.3 ml. With a Cavro XD 1000 pump, the maximum syringe size is 5 ml. The operation requires a two-dispensing cycle with a buffer volume exceeding 5 ml. The dispensing rate for this fixation is sufficient to mix the red blood cells and mix the buffer, ferrofluid and capture enhancing reagent.

  At station 4, dispense 3 ml of wash solution. This consists of a combination of wash dilution buffer and system buffer to 3 ml. Station 4 is a resuspension dispense where the fluid flow from the probe (51) is directed to the collection cells on the wall of the sample tube.

  Station 5 dispenses a final volume of 650 uL. At station 7, add a 650-800 uL mixture of system buffer and wash dilution buffer. After this dispensing, cell sedimentation for ferrofluid reduction begins.

  Station 9 dispenses 150 ul of stable reagent after removal of unbound ferrofluid. The system may perform aspiration and dispensing as a mixing step after the initial dispensing to ensure that the cells are uniformly suspended in the solution.

  The moving probe (51) is narrowed to 1.27 mm for 40 mm so that the probe tip enters the 2.33 mm diameter opening of the cartridge and reaches the bottom of the 30 mm analysis chamber. The moving probe (51) teflons the outer surface to minimize cell carryover. The migration probe (51) wash sequence is designed so that the carryover details are less than 2 cells in 50,000.

Sensor System Design Overview Further improvements are found in the design of sensor systems used to verify process integrity. Processing steps include protoplast aspiration, magnetic separation, post-separation aspiration, reagent addition and final transfer to cartridge, among others. Since the most serious error that can be made is processing the sample incorrectly, the system needs to automatically verify that these steps were performed properly. In some cases, otherwise, incorrect cell capture or incorrect cell labeling will result in an abnormal end result for the sample, such as an incorrect cell count. In other cases, improperly performed steps lead to biohazard conditions in the device such as aspiration failure after reagent addition and loss of one or more samples. A sensor system is used to verify that all samples have been processed correctly. Once the reagent is added to the sample, the sample is involved until completion.

Bulk fluid sensing System fluid is used in the system to prepare and clean the probe (51). It may also be used as a backing fluid for the probe tube and as the reagent itself. As described in the previous section, the sensor is used to verify that there is sufficient system fluid at the start of the run to complete the run. Losing fluid during a run loses proper priming maintenance of the fluid-supported probe (51). This results in an inaccurate reagent volume and the probe (51) cannot be washed properly.

  Analog sensing is used to periodically measure the weight of the bottle during device operation. The software predicts the amount of fluid required during a run based on general priming requirements, test definition information and the number of samples run. With the start of the run, the system determines if it is sufficient to complete the run. Otherwise, the run cannot be made until the operator refills the fluid.

  The system predicts worst case fluid usage based on the batch size entered by the user and multiplies by the number of samples run by calculations including initial system priming and probe wash.

Waste sensing There are additional sensing requirements for liquid waste. Analog sensing is similar to that described for system fluid sensing. Here, a sensor is used to verify that there is sufficient capacity at the start of the run and that the bottle does not fill up during the run. Optionally, a liquid waste line to the laboratory drain can be used. The capacity limit of the waste bottle for liquid waste includes waste from probe priming and cleaning, and liquid waste aspirated from the sample tube. The waste bottle itself is sized to accommodate waste for multiple batches of samples.

  Sensors are used to mitigate the loss of liquid waste capacity, in which case probe priming, probe cleaning and general aspiration are stopped and the run is aborted. The sensor is used during maintenance activities such as daily cleaning procedures.

Data buttons and barcode sensing devices act both as a sample cartridge holder and as a device that introduces a magnetic field into the sample when dispensed into the cartridge. With respect to the sample cartridge element, the cartridge is loaded into the machine. Using the data button located on the cartridge holder, the information written by the system software about the sample is transferred to the imaging system. The data button has a unique ID for traceability.

  The cartridges themselves are ordered and have a barcode that allows the system to identify the cartridge ID. In order to detect that a cartridge is installed in the cartridge holder, the device reads the barcode. The system provides an access door for the carousel for safety and prevents operator access while the carousel is moving.

  The cartridge carousel has 8 positions. The cartridge / holder is placed at the operator access position, at the data button / barcode reading position, and at the moving probe position (station 9). The system must know that the correct cartridge is in place. The carousel utilizes a sensor sequence that identifies each position based on absolute criteria.

  When a cartridge / holder is loaded into a cartridge carousel, its presence is confirmed by an embedded data button. The system verifies that the cartridge is new and not already in use by the device by comparing the serial number with the device database of the previously scanned cartridge.

  Use the data button embedded in the cartridge / holder to transfer data about the sample to the imaging platform. Data is written to the cartridge / holder at the time of sample dispensing to the cartridge. Typical data includes sample information, reagent lot information, tests performed, rejection information, and other relevant information. A data button connection is created for each cartridge / holder.

Reagent Pack Sensing The primary reagent is loaded into the device in a single reagent pack (52). The reagent pack area is in the process ring. The probe moves over the ring and reagent pack. The pack contains one bottle for wash dilution buffer and six small bottles for other reagents required for processing. The pack design is intended to place the bottles in a radial fashion with respect to probe access during system operation. During a batch run, the reagent carousel places an appropriate bottle at each probe (51) station when needed for reagent aspiration. The software schedules probe access to the sought bottles in order to maintain the position of the reagent carrier and serve all probes simultaneously.

  The reagent carousel places the reagent in and out of the reagent at one or more load positions relative to the operator. During the run, the carousel (52) must place the appropriate reagent bottle at the probe (51) station for reagent aspiration and the correct reagent bottle must be located at the probe (51) for reagent access. And The sensor is designed to allow absolute reagent pack placement. The reagent data buttons described above are not limited to lot numbers, expiration dates, etc. for use in inventory control, but contain general information about such reagents. As the reagent is aspirated, the data button reduces the amount of reagent left in the pack. In this manner, if the pack is removed from the device for any reason (ie, refrigerated), the device will know the remaining amount the next time the pack is viewed. Thus, the data button may be used for lot control, on-board time tracking and expiration.

Red Blood Cell Sensing As described above, the operator loads a tube with a directly centrifuged blood sample into the process ring. Each entry requires the operator to press a button so that the system directs the ring to the next position. This process is repeated for the number of tubes indicated by the operator. The system reads the barcode label on the tube and detects the red blood cell layer position in each sample tube before loading the next tube. For this process, the barcode reader is placed in the next position behind the tube loading position. A CCD camera is used to sense the red blood cell layer in the sample. The system uses barcode labels and / or the presence of red blood cells as a means for tube detection. The system will not process the batch of samples if the red blood cell layer of the sample is not detected.

  The location of the red blood cell to protoplast transition varies depending on the patient hematocrit value and the original sample volume. Therefore, it is important to measure the position of the red blood cell layer in order to know how much protoplasm is removed. Normal hematocrit levels range from 45 to 52% for adult men and 37 to 48% for adult women. Assuming a range of hematocrit values of 20-60% for a 7.5 ml sample, a physical range of 1.5 to 4.5 ml of red blood cells in a 7.5 ml centrifuged whole blood sample.

  The system removes everything except about 0.5 ml of protoplasm without disturbing the cells in the buffy coat. To do this, erythrocytes that absorb light in the 532 nm region are illuminated with green LED light (532 nm). A CCD camera captures an image of the illuminated tube. Under these light conditions, the boundary of the erythrocyte layer has a high contrast with that of the plasma layer. An image analysis algorithm is applied to the image to determine where red blood cell and protoplasmic metastasis occurs.

One embodiment of the algorithm in the present invention involves the calculation of pixel intensity to locate the red blood cell layer after image capture based on the following steps:
1—Create the first derivative by subtracting the previous pixel value from the current pixel value.
2- Make the second derivative by subtracting the previous pixel value from the current pixel value.
3- Average five pixel collections by averaging the sample and its four closest approximations.
4- Plot the original data, first and second derivatives.
5-The position of the maximum zero crossing of the second function is the position of the red blood cell layer.

  The location of the transition is used for protoplast aspiration. The system senses the transition between protoplasm and red blood cells with an accuracy of +/- 0.25 mm.

Pump Sensing Syringe Pump Syringe pumps include error detection to sense improper movement of the piston resulting in an incorrect amount of reagent being aspirated and dispensed. In addition, the syringe pump includes overload detection. The syringe pump includes an integrated valve with the sensor to detect errors associated with valve operation.

Peristaltic pumps Peristaltic pumps include error detection for such faults, but are not limited to such things as pump heads not moving or pump heads moving in the wrong direction. This provides only partial protection against aspiration failure. For example, a failed pinch valve or pump tube will still cause a visual failure. The aforementioned fluid sensing is designed to mitigate failure modes that the pump cannot detect.

Fluid Level Sensing An important sensing function is to ensure that the appropriate reagent volume is added to the tube. After all reagent addition, fluid level sensing is performed to confirm that the appropriate volume of reagent has been added from the previous step.

  The reagent probe (51) is used to aspirate fluid from the reagent bottle and dispense them into the sample tube. The reagent probe (51) of the station 9 is a “moving probe” that transfers the final product to the cartridge. At most stations, the reagent probe (51) performs a “resuspension” dispense to resuspend the magnetically collected cells on the wall of the tube.

The main purpose of reagent probe level sensing is to detect the presence of fluid in the accessed reagent bottle and in the sample tube after the reagent has been dispensed. A typical reagent bottle aspiration sequence is:
a. Place the reagent bottle on the probe station,
b. Search for fluid
c. Aspirate the required volume while moving the probe (51) downward to follow the fluid level.
d. Search for fluid after aspiration
e. Calculate fluid level changes to determine if reagent collection was successful.

Magnet position sensing The magnet shuttle places the magnet close to the tube during separation and magnetic incubation. The magnet is withdrawn from the tube during cell resuspension dispensing and when indexing the process ring. The magnet shuttle meshes with the tube rotation motor. Engagement with the tube rotation motor occurs at different magnet shuttle positions. For example, at stations 2 and 3, motor engagement occurs when the magnet shuttle is pulled apart. At station 4, motor engagement occurs when the magnet is pulled. The meshing mechanism allows the meshing position to be adjusted. The mechanism includes a home sensor that is activated when the magnet shuttle is retracted. The system monitors the motor step count relative to the home position to ensure proper magnet position during processing.

Tube Rotation Sensing During magnetic incubation and separation, tube rotation is necessary to fully collect labeled cells and place them for subsequent resuspension after aspiration after separation. The device senses that the tube lid is rotating at the bottom of the tube holder by means of an optical sensor and a flag array. The spring loading force of the tube holder with the knurled features on the tube holder cup and the features of the tube itself creates a resistance sufficient to verify tube rotation.

Magnet design Figure 7 shows the sample and associated magnet design at each station (1-9).

  The probe is constructed from an Inconel alloy rather than stainless steel because of the nonmagnetic properties of Inconel. Inconel is a non-magnetic, corrosion and oxidation resistant, nickel-based alloy typically used for heat shields, furnace hardware, and gas turbine engine ducts. Inconel has a magnetic permeability of 1.000 and is therefore not magnetic in the presence of a strong magnetic field gradient.

  Station 2 and 3-magnetic incubation. With respect to stations 2 and 3, the purpose of the magnet (72) is to pull the ferrofluid particles across the diameter of the tube (71) to improve the collision between the target cells and the magnetic particles. The magnetic field causes the particles to form “chains” as they pass through the sample. This further improves collisions and subsequent cell attachment. The incubation magnet (72) used for stations 2 and 3 is in a tripole format with a 70 degree angle between the north-south-north or south-north-south magnet.

  The magnet arrangement is an improvement over the previously described quadrupole arrangement (US Pat. Nos. 5,186,827 and 5,466,574). Since the magnet (72) is required to be attracted and pulled away from the sample, the magnet has an open side that allows the sample to be placed in and out of the magnetic field. In the aforementioned quadrupole arrangement, the sample tube needs to be removed vertically from the magnetism. This makes automation difficult.

Station 4-Separation Station 4 magnetic separation is designed to separate ferrofluid-bound cells from a sample and concentrate the cells in a location resuspended by a 3 ml fluid stream. The station 4 magnet (73) is designed as a deflection dipole magnet with a 180 degree angle between the north and south facing magnets. The tilt of the magnet is adapted to the angle of the cone portion of the sample tube to provide both a gradient vector towards the side of the tube and a gradient vector towards the bottom of the tube. This arrangement is designed to bring cells to the wall of the tube, below the 3 ml mark on the tube, and subsequently allow resuspension of the cell after aspiration. In this arrangement, an access gradient of 20 K Gauss / cm exists inside the wall of the tube. This improvement is superior to the gradient gradient found in previous quadrupole designs.

Station 5-Magnetic Wash The purpose of the Station 5 magnet is to separate the magnetically labeled cells resuspended in 3 ml protein-based buffer at Station 4. The magnet (74), in conjunction with the tube rotation, serves to place the cells at specific locations on the wall of the tube before resuspending the permeation reagent (US 10 / 780,349) and staining reagent and cells. As with the station 4, the magnet design for the station 5 is an inclined two-pole arrangement. Here, the magnet (74) is shorter due to the lower sample volume. The magnet (74) is tilted to provide both horizontal and downward magnetic forces on the cells as the cells are resuspended in a volume of less than 3 ml. The resuspension volume is between 650 and 800 ul.

Station 7-Magnetic Cleaning The magnet design for station 7 is similar to the magnet design for station 5. The size of the magnet (75) is shorter than that used at station 5 due to the reduced reagent dispensing volume.

Station 9-Ferrofluidic fractionation The purpose of the magnet design for station 9 is to capture the sedimented cells in the sample tube (at station 8) for about 20 minutes. The density of the fluid at this stage is expected to be about 1.01 gm / ml (note: cells are expected to have a density between 1.06 and 1.08 gm / ml). Captured at a point above the bottom of the bottom. This allows the probe (51) to aspirate all residual liquid containing unbound ferrofluid and other reagents while leaving the cells intact.

  For station 9, the magnet design is a dipole with a 180 degree angle between the poles. The shape of the magnet (76) allows contact with the outside of the tube at a predetermined vertical position. The magnetic field strength is sufficient to attract and hold cells near the pole, but not so strong as to attract unbound ferrofluid in solution (note: place the tube near the bottom of the tube) All metal elements used to maintain the same must be non-magnetic). Along with protocols for control of sedimentation time, magnet engagement time, suction height and suction speed, the magnet is optimized for ferrofluid removal and minimal cell loss.

Computer Interface The host computer hardware interface to the device is designed for easier operation. The keyboard is less than 18 ”(46 cm) and has enough space to support other languages in addition to English. The function keys have at least 10 function keys along the top. Can be easily stored under the device. The display is a flat panel, SVGA Color with a viewing angle of 60 degrees or more. The display embedded in the front of the device has an 8.5 "diagonal size. Six buttons are attached along the bottom of the display. Any mouse can be used during device development or service calls. The device has an IDE hard drive with 20 GB or more storage. The integrated RW CDROM drive has functions for system and software update, backup and repair functions. The printer is used for run reports, calibration lists, etc. The printer uses 3 "thermal paper with cutting features. Four serial ports are available; one for reading barcodes, one for external modems, and two for data button readers Yes, use a door sensor to verify that the door is closed before starting a run or cleaning procedure.Use the sensor to detect the opening of the process area door.The sensor uses firmware and software. Monitored by.

  The host computer regularly monitors the temperature in the internal area with an accuracy of +/- 1 degree Celsius. Of the two sensors used, one is inside the sample processing area and the other evaluates a ventilator for electronics.

Reagent packaging Reagent packaging has been developed to support fast and error-free instrument design. The bulk fluid includes the system support fluid used for system cleaning and the diluted bleach solution. A large volume of solution is filled with a specific bulk reagent or liquid according to the instructions. Large volumes of containers are designed to be reusable with instructions for regular bottle cleaning. The reagent pack consists of a disposable reagent holder, a reagent bottle, an integrated data button and a reusable reagent pack carrier supplied by the device. The reagent bottle is an industry standard bottle but can be replaced with a custom bottle of similar shape and size. The reagent pack carrier is designed to fit into the carrier and subsequently be placed on the device. The carrier can be reused after the reagent pack is exhausted. The carrier also provides electrical coupling for ground coupling required for sensing the volume level of individual bottles in the reagent pack and for data button coupling for reading and writing to the reagent pack data buttons. The carrier provides a means for maintaining individual bottles and allows the lid to be removed with only one hand of the operator. In addition, the evaporative cover can be optionally applied to a reagent pack in a reusable carrier for storage in a refrigerator during use.

System Cleaning To prevent biological growth and protein accumulation that occurs in most liquid-based chemical devices, system designs are enforced to facilitate automatic periodic cleaning. The recommended cleaning solution is a bleach solution (5% sodium hypochlorite) commonly found in laboratories. The automatic cleaning procedure is defined by:
1- Prime the system thoroughly with cleaning solution.
2- Give the cleaning agent a residence time to clean the tube.
3- Dry the system and remove as much cleaning solution as possible.
4- Flush dry lines with deionized water or system fluid.
5- Reprime the system with normal system fluid.

  The entire cleaning system is designed to be a “walk away operation”, which differs from the cleaning procedure of the prior invention in that, once initiated, the device of the present invention does not require operator intervention. It means that it is not necessary at all.

  These improvements only reflect the many possible applications of the apparatus and methods that can be anticipated by one skilled in the art and are therefore not intended to limit the scope of the invention in any way. Should be understood and recognized. Accordingly, other objects and advantages of the invention will be apparent to those skilled in the art from the detailed description, together with the associated claims.

Claims (4)

a. obtaining images of the plasma and red blood cell layers;
b. Obtain the first derivative by subtracting the previous pixel intensity value from the current pixel intensity value;
c. obtaining the second derivative by subtracting the second previous pixel intensity value from the second current pixel intensity value;
d. average 5 pixel ensembles, where the pixel and its 4 nearest neighbors are pooled;
e. Show the mean data, the first derivative, and the second derivative in coordinates;
f. indicates the location of the maximum zero tolerance of the second derivative, where the location is the red blood cell layer, and represents the contact surface between the red blood cell layer and the protoplasmic layer in a distributed blood sample. Algorithm to evaluate.   The algorithm of claim 1, wherein the evaluation is used to remove the plasma layer for automatic enrichment of target cells.   The algorithm of claim 1, wherein the target cells are selected from the group consisting of epithelial cells, endothelial cells, fungal cells, bacterial cells, cell debris, cellular components, and combinations thereof.   The algorithm of claim 1, wherein the processing is under the preparation of diagnostic image analysis, diagnostic nucleic acid analysis, and combinations thereof.

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