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EP-4208706-B1 - AUTOMATED METHOD FOR DIRECT SAMPLING OF IMMUNE CELLS FROM WHOLE BLOOD OR OTHER BIOLOGICAL SAMPLES IN MICROWELL PLATES

EP4208706B1EP 4208706 B1EP4208706 B1EP 4208706B1EP-4208706-B1

Inventors

  • LIU, ZHAOPING
  • BARNES, STEPHEN
  • PEREZ, DOMINIQUE
  • SUSKI, CHRISTOPHER

Dates

Publication Date
20260506
Application Date
20210728

Claims (15)

  1. A method for automatic sampling of immune cells from a biological fluid sample (12) deposited in a well (10) of a microwell plate (100), the well (10) having a wall, the biological fluid sample containing red blood cells (RBCs) (15) and magnetic beads (14) which are designed to bind to the RBCs (15) in the sample (12), comprising the steps of: a) placing the microwell plate (100) on a shaker (104) having a magnetic adapter (102) including at least one magnet (120), wherein the magnet (120) causes the RBCs (15) bound to the magnetic beads (14) to be held against the wall of the well (10); b) shaking with the shaker (104) the microwell plate (100) in a manner and for a time period so as to suspend substantially evenly the immune cells (17) in the biological fluid sample within a region of the well (10) such that the immune cells (17) are substantially isolated from the RBCs (15) held against the wall of the well (10); and c) lowering a sample probe (200) into the well (10) in the region of the well (10) and withdrawing a portion of the sample containing the immune cells (17) from the region, wherein the shaker (104) further comprises a home position sensor, and wherein the method further comprises the step of shielding the home position sensor from a magnetic field created by the magnet (120).
  2. The method of claim 1, wherein the magnetic adapter (102) comprises a structure holding individual magnets (120), one for each well (10) of the microwell plate (100), wherein the magnetic adapter (102) is configured to fit onto an upper surface (106) of the shaker (104) and be sandwiched between the upper surface (106) and the microwell plate (100), and wherein the magnets (120) cause the RBCs (15) to be held to a bottom wall (16) or a side wall (19) of the well (10).
  3. The method of claim 1 or claim 2, wherein the biological fluid comprises a cyst fluid sample, amniotic fluid, a bone marrow sample, a cerebrospinal fluid sample, a liquid biopsy, a chorionic villus sample, or whole blood.
  4. The method of any of claims 1 to 3, wherein: i) the sample probe (200) is part of a flow cytometer (400), and wherein the method includes step d) of introducing the portion of the sample containing the immune cells (17) into the flow cytometer (400) and/or ii) the shaking step comprises the step of operating the shaker (104) in an eccentric rotation at a speed of between 100 rpm and 1500 rpm, and/or iii) the magnetic beads (14) comprise magnetic or paramagnetic beads with an estimated size between 1 nm - 50 um, conjugated with a specific molecule that binds to a molecule expressed on the RBCs (15) but not the immune cells (17).
  5. The method of any of claims 1 to 4, wherein: i) in step a) and c) the shaker (104) is in a non-shaking condition, and/or ii) the magnetic adapter (102) including the at least one magnet (120) is integrated into the shaker (104), and/or iii) the method further comprises the step of placing a pierceable seal over the microwell plate (100) prior to performing step a), and/or iv) the sample further includes in-well marker beads or in-well counting beads.
  6. A flow cytometer (400), comprising: a robotic sampling probe (200); a shaker (104) having a top surface (106); a magnetic adapter (102) configured to be placed on the top surface (106) of the shaker (104), the magnetic adapter (102) having one or more features for holding a microwell plate (100) placed thereon, the microwell plate (100) having a plurality of wells (10) with biological fluid sample, wherein the magnetic adapter (102) cooperates with structures on the shaker (104) so as to be removably fitted to the top surface (106) of the shaker (104), wherein the magnetic adapter (102) comprises a structure holding one or more magnets (120), and wherein the magnetic adapter (102) is configured to be sandwiched between the shaker (104) and the microwell plate (100); a control system configured for shaking the shaker (104) with the microwell plate (100) in a manner and for a time period so as to suspend substantially evenly immune cells (17) within a region of the well (10) of the microwell plate (100), wherein the immune cells (17) in the region of the well (10) are substantially isolated from red blood cells (15) magnetically bound to a wall of the well (10); and analytical instrumentation for conducting one or more measurements on immune cells (17) withdrawn from the well (10) of the microwell plate (100), wherein the robotic sampling probe (200) withdraws the immune cells (17) from the well (10) and introduces the immune cells (17) into the instrumentation, and wherein the shaker (104) further comprises a home position sensor, and wherein the shaker (104) further comprises a shield (302) positioned between the shaker (104) and the magnetic adapter (102) shielding the home position sensor from a magnetic field created by the magnets (120).
  7. The flow cytometer of claim 6, wherein the biological fluid sample comprises a cyst fluid sample, amniotic fluid, a bone marrow sample, a cerebrospinal fluid sample, a liquid biopsy, or a chorionic villus sample, and/or wherein the biological fluid sample comprises whole blood.
  8. The flow cytometer of any of claims 6 to 7, wherein the control system operates the shaker (104) in an eccentric rotation at a speed of between 100 rpm and 1500 rpm.
  9. The flow cytometer of claim 8 wherein the red blood cells (15) are bound to magnetic beads (14) with an estimated size between 1 nm - 50 um, conjugated with a specific molecule that binds to a molecule expressed on a cell surface of the red blood cells but not a cell surface of the immune cells (17).
  10. The flow cytometer of any of claims 6 to 9, wherein the sampling probe (200) withdraws a sample from the well (10) of the microwell plate (100) after the shaking of the microwell plate (100) and while the immune cells (17) are suspended substantially evenly in the region of the well (10) and optionally wherein the withdrawal of the sample from the well (10) occurs at a sampling time of between 0.5 second and 5 minutes per well (10).
  11. The flow cytometer of any of claims 6 to 10, further comprising one or more reagents for the biological fluid sample, wherein the one or more reagents include magnetic beads conjugated to an antibody designed to bind selectively to the surface of RBCs (15).
  12. The flow cytometer of any of claims 6 to 11, wherein the magnetic adapter (102) cooperates with structures on the shaker so as to be removably fitted to the top surface of the shaker, the magnetic adapter (102) comprising a structure holding an array of one or more magnets, and wherein the magnetic adapter is configured to fit onto the upper surface of the shaker and be sandwiched between the upper surface of the shaker and the plate.
  13. The flow cytometer of claim 12, wherein the magnets (120) are arranged in the magnetic adapter (102) so as to be in registry with a bottom portion of the wells (10) when the microwell plate (100) is placed on a top surface of the magnetic adapter (102).
  14. The flow cytometer of any of claims 12 to 13, wherein the robotic sampling probe (200) is controlled to move in X, Y and Z directions relative to the microwell plate (100).
  15. The flow cytometer of any of claims 12 to 14, wherein the shield (302) is a ferromagnetic shield (302) positioned between the top surface (106) of the shaker (104) and the magnetic adapter (102) and/or wherein: a) the magnetic adapter (102) includes an array of individual magnets (120), one for each well (10) of the microwell plate (100), or b) the magnetic adapter (102) is configured to position the magnets (120) in a space proximate to a side wall (19) of the wells (10) of the microwell plate (100).

Description

Cross-Reference to Related Application This application claims priority to United States Non-Provisional Patent Application No. 17/009,225, filed September 1, 2020. Background This disclosure relates to a method for direct sampling of cells, such as immune cells (lymphocytes, neutrophils, monocytes, and macrophages, or generally leukocytes or white blood cells (WBCs)), from biological fluid samples which are loaded into the wells of a microwell plate. One particular application for the present method is a direct sampling of immune cells from whole blood samples. The method is also suitable for direct sampling of immune cells from other biological fluid samples which may contain red blood cells (RBCs), such as a cyst fluid sample, amniotic fluid, a bone marrow sample, or a cerebrospinal fluid sample. The term "microwell plate" is used to refer to a test device format in the form of a flat plate forming an array of many small individual sample-holding wells, typically 6, 12, 24, 48, 96, 384 or more wells per plate, or to refer to a test device format in the form of a test tube array, typically 40 test tubes in an array. The term is sometimes referred to in the art as a "microtiter plate" or "microplate." Such microwell plates are typically used in conjunction with a sample processing apparatus, which automatically extracts a portion of the sample from one of the wells and introduces the sample into an analytical instrument, for example a flow cytometer, hematology analyzer, cell sorter, mass spectrometer, etc. which conducts one or more measurements of the extracted sample. Sampling whole blood with a cytometer for white blood cells (WBCs) analysis is difficult because whole blood tends to clog small flow paths. Also, it is known that populations of WBCs and RBCs are difficult to distinguish on a traditional cytometer. Hence, the art has developed methods for removing RBCs from a whole blood sample. One method, red blood cell (RBC) lysis, uses a buffer solution such as ammonium chloride, which lyses RBCs with minimal effect on leukocytes. The use of traditional RBC lysis methods in microplate format used for whole blood sample processing is labor intensive, it creates RBC debris that can clog the cytometer flow cell, makes the cytometer very dirty, and significantly increases the carryover from one sample to another. Further, the RBC lysis method may cause a loss of data integrity since the hypotonic buffer used in the lysis is not physiological and may affect the normal immune cell activity. Another method, traditional gradient centrifugation, can be used for purification of WBCs and this method is used for samples in a test tube format, but it is not applicable for a microplate format. Hence there is a need in the art for a method of automatically sampling immune cells from samples containing RBCs or other biological fluid samples in a microwell plate format. US 6689615 B1 and US 9063044 B2 disclose prior art methods of automatically sampling immune cells from samples containing RBCs in a microwell plate format. Summary The subject of the invention is defined by the appended claims. According to one embodiment useful for understanding the invention, a method is provided for automatic sampling of cells, such as immune cells, from a biological fluid sample deposited in a well of a microwell plate, the well having a wall (i.e., a bottom wall or a side wall). The sample contains, for example, (1) RBCs and (2) magnetic beads which are conjugated to antibodies or otherwise designed to bind to RBCs in the sample. The method includes steps of: a) placing the microwell plate on a shaker having a magnetic adapter including a magnet, wherein the magnet causes the RBCs bound to the magnetic beads to be attracted to and migrate to the wall of the well and be held against the wall; b) shaking with the shaker the microwell plate in a manner and for a time period so as to suspend substantially evenly or homogeneously the immune cells in the biological fluid sample within a region of the well but still retain the holding of the RBCs to the wall of the well such that the immune cells are isolated from the RBCs in the region of the well; and c) lowering a sample probe into the well in the region of the well and withdrawing a portion of the sample containing the immune cells from the region. In another embodiment useful for understanding the invention, a shaker system is described in the form of a shaker having a top surface that is configured for shaking a microwell plate in a controlled and programmable manner. The shaker includes a magnetic adapter cooperating with structures on the shaker so as to be removably fitted to the top surface of the shaker. The magnetic adapter is in the form of a substantially flat structure holding an array of individual magnets, and wherein the magnetic adapter is configured to fit onto the top surface of the shaker and be sandwiched between the top surface of the shaker and the microwell plat