MXPA96004813A - Method of selection of a population or subpopulation of a sample - Google Patents

Abstract

A separation procedure for separating a selected desired or undesired population from a biológical sample utilizing relatively heavy, dense particles and gravity sedimentation. The particles have one or more reactants bound thereto which are specific to and will bind with the selected population. The particles preferably are mixed with the sample by repeatedly causing the particles to settle through a substantial portion of the sample to bind to the selected population. The particles with the bound selected population then are allowed to preferentially settle in the sample and the supernatant including the non-selected population is separated from the particles with the selected population bound thereto. The particles can be heated for sterilization and endotoxin removal.

Description

METHOD OF SELECTION OF A POPULATION OR SUBPOBLATION OF A SAMPLE Technical Field The present invention relates generally to the separation of a desired or unwanted population or subpopulation from a sample, to obtain the desired population or subpopulation alone, or an improved population or subpopulation, eliminating one or more undesired subpopulations thereof. More particularly, the invention relates to separating the desired or unwanted population or subpopulation, such as from bone marrow or blood cells, by linking the population or subpopulation to relatively dense particles, and the use of sedimentation by severity to separate the population or subpopulation of the supernatant from the remaining sample. F'. The improvement of a population or a subpopulation of a sample can be used for many types of applications. For example, in the bone marrow, the removal of all non-Jodgkin's B-cell lymphoma cells may be desired in the case of a B-cell lymphoma. If the bone marrow is to be purged from the B cells, and reintroduce the patient, it is important that the bone marrow be completely purged, and that the bone marrow not be otherwise damaged.

BACKGROUND ART At present, one approach is to use a plurality of magnetic microspheres, typically formed of a magnetic material based on polymer of a relatively low density. It is desired that the microspheres be of a relatively low density, because the microspheres are mixed with bone marrow or blood, and are specifically designed not to settle by gravity sedimentation. The microspheres are typically of a small size, generally about or less than one miera - diameter. However, a product sold by Dynal, Inc. of Great Neck, N.Y., uses magnetic polymer microspheres having a nominal diameter of 2.8 or 4.5 microns, with a low microsapheres density of the order of 1.5 grams / cubic centimeter. It is intended that the magnetic microspheres of the prior art be kept in suspension in the sample, and, consequently, be designed for a very slow or substantial removal of settlement by gravity in the sample suspension. The magnetic microspheres have at least one antibody bound thereto, specific for the population or subpopulation that it is desired to remove. Frequently, as in the Dynal process, a first monoclonal antibody binds to the cells of interest, and a second antibody specific for the first monoclonal antibody binds to the microspheres. The cells are typically isolated from whole blood or bone marrow, and then washed before binding the monoclonal antibody thereto, whose washing step causes a non-discriminating loss of cells. The microspheres and the cells are then mixed together to bind the microspheres to the cells by means of the first and second antibodies. To purge the blood or bone marrow, a sample would be mixed with a plurality of the microspheres bound with antibody, and then placed in a magnetic field. The remaining sample or supernatant is removed while the microspheres are maintained in the magnetic field. This procedure should typically be repeated, since a single purge step will generally not deplete a sufficient percentage of unwanted populations or subpopulations. The goal of purging is to eliminate all (100 percent) of the population or objective subpopulation. This is generally not feasible, and the sample is purged as close to 100 percent (100%) as is feasible. The magnetic removal procedure presents several problems. The method also removes a number of cells not specifically from other populations during each step of elimination. This decreases the yield, that is, the percentage of the desired population remaining. A single step of elimination results in a variable yield of a relatively low percentage, also reducing each successive step the performance. In addition, magnetic microspheres are relatively expensive. The magnetic microsphere procedure has also been used to improve a subpopulation for the study of the subpopulation. In this case, the magnetic microspheres are linked to the desired subpopulation, and the microspheres with the cells bound thereto, are removed from the sample. Then the subpopulation can be studied directly, or it can be removed from the microspheres for study. This procedure is delayed, in the order of approximately one (1) to six (6) hours or longer, and, arguably, does not result in a native subpopulation, since the subpopulation has had at least one monoclonal antibody linked to at least a type of cellular antigen. An additional use for purging is the study / improvement of a specific subpopulation, such as the CD4 population of lymphocytes (L). In this case, the microspheres have monoclonal antibodies linked to them, specific for one or more non-CD4 populations. The elimination of the other populations increases the number of CD4 cells in the total remaining cell population of the sample. However, when the magnetic microspheres are used, the non-specific removal of a portion of the CD4 subpopulation can seriously affect the remaining number of the CD4 subpopulation. The non-specific elimination of cells * can become more of a problem when a large volume of sample is used, such as five (5) milliliters and larger, the volume of which then requires a large number of magnetic microspheres. When the magnetic microspheres are then placed in a magnetic field, there is often a non-specific trap and removal of other non-objective cells. Other methods of positive or negative selection, including surfaces labeled with antibodies, have been employed to generate subpopulations of cells from a mixture of different cell types. These methods usually have an antibody covalently bound to a plastic surface or to the polymer particles in a column. In general, the population of mixed cells is combined with the bound antibody, either by adding them to a column and allowing them to incubate, or by letting them settle on a surface. These procedures work optimally when initially the red blood cells (RBCs) and the plasma of the population of mixed cells have been eliminated by the preparation of a polished coating or a mononuclear preparation by density gradients, washing the cells and combining them with the surface labeled with antibody. Both methods also require the preparation of the separation and washing system with a pH regulator before use, which, with incubation times of thirty to sixty (30-60) minutes with the antibody, results in a procedure that It takes a minimum of three hours for the column and flask method.These methods can be used either for negative selection or for positive selection of the population of cells of interest.In both methods, direct separation results in a population highly enriched, with the resulting loss of non-target cells in a non-specific manner.The cells released can have antibody on the cell surface, and are often activated by the separation technique, which is often not desirable. which incorporate the invention, can be used with a variety of immunological reactions, such as immunological reactions involving r EACTIVES AND CELLS As used herein, cells are defined as cells of animals or plants, including cell bacteria, fungi, which can be identified separately or in aggregates. Cells are the minimum structural aggregate of living matter capable of functioning as an independent unit. For example, the cells may be human populations of red blood cells and white blood cells (GBS), cancer cells or other abnormal cells of tissue, bone marrow, and / or blood samples. It can be expected that properly labeled or labeled cells will be operated by the method and apparatus of the invention in the same manner as examples of human blood or bone marrow cells. As used herein, the term "reactive" defines different molecules, such as monoclonal or polyclonal antibodies, that detect and react with one or more specific complementary molecules, such as antigens, that are on the surface of a cell. Some examples are given immediately: Reagent Specific Molecule Antibody Antigen Drug Drug receptor Hormone Hormone receptor Growth factor Receptor growth factor. Lectin Molecule of carbohydrate Nucleic acid sequence Complementary nucleic acid sequence Enzyme Cofactor or inhibitor.

The reagents are coupled or bound to specific molecules on the cells. It would be desirable to have an effective method to purge or select one or more subpopulations without affecting the remaining oblations of a sample, such as whole blood or bone marrow. The method must be economical, fast, should result in high performance, and should not be restricted in the volume of the sample on which it is going to act. Description of the Invention The invention provides a method and apparatus for separating a desired or unwanted population or subpopulation from a sample of biological fluid, such as whole blood, in a rapid manner and with high performance. A plurality of dense, relatively heavy particles are mixed, having one or more reagents, such as monoclonal or polyclonal antibodies, linked thereto, with the sample. The antibodies bound to the particles can be directed towards cells that are not of interest. The particles with the cells bound to them are allowed to settle differentially by gravity, and then the remaining sample is removed, this increases the number of remaining cells of interest in the sample that were not objective of the particles. The antibodies bound to the particles can also be directed towards the cells of interest. The rest of the sample fluid and non-objective cells can then be removed from the particles, with the target cells bound to them, and analyzed to determine how many non-objective cells were removed.

Then the objective cells of the particles can also be removed for further analysis. A preferred particle material of interest may be nickel. The nickel particle can be heated to sterilize the particle when desired. If the sample has been purged and is going to be transplanted to a human being, a magnetic field and a washing procedure can be used to remove the red blood cells, and also ensure that all the dense particles in the sample have been removed. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic block diagram of a first embodiment of a selection method in accordance with the present invention. Figure 2 is a conceptual embodiment of a particle with objective cells linked thereto, in accordance with the present invention. Figures 3A-3E are front, side, and end views of a blood bag mixer of the present invention. Figures 4A and 4B are control histograms of whole blood and enriched with granulocytes obtained in accordance with the present invention. Figures 5A and 5B are control histograms of whole blood and enriched lymphocytes, obtained in accordance with the present invention.

Figures 6A-6E are histograms comparing the gravity settlement of the Rhone-Poulenc magnetic particles with the dense particles of the present invention. Figures 7A and 7B are control histograms of whole and depleted blood of platelets and granulocytes, obtained in accordance with the present invention. Figures 8A-8C are control histograms of whole and depleted blood of platelets and granulocytes, obtained in accordance with the present invention. Figures 9A-9F are histograms illustrating the results of the gravity settlement of the present invention, compared to the accelerated settlement of the present invention. Figures 10A-10F are histograms illustrating the results of varying the settling times by gravity of the present invention. Figures 11A-11C are histograms comparing the heated particles with the unheated particles of the present invention. Modes for Carrying Out the Invention Now referring to Figure 1, a first embodiment of a method and apparatus for selection of conformity "" in the invention, is designated in general by reference numeral 10. The selection apparatus 10 includes a fluid sample 12 containing a previously selected population or subpopulation that The population or subpopulation can be a population or subpopulation of cells, including: cells found in the bone marrow or in the blood, such as platelets, neutrophils (N), eosinophils (E), monocytes (M), lymphocytes (L), subsets of lymphocytes, immature cells from embryonic stem cells for mature leukocytes, and diseased cells, such as human or animal cancer cells. be a biological fluid, including whole blood or a portion thereof, bone marrow, spinal fluid or urine, or other fluids containing populations or subpopulations, such as described above. The separation apparatus 10 also includes a source of particles 14. The particles 14 include a monoclonal or polyclonal antibody linked thereto, which will specifically bind to the selected cells. The antibody can be bound to the particles 14 directly, either covalently or by adsorption, or indirectly by means of a second antibody in any conventional manner. A plurality of the particles 14 and at least a portion of the sample 12 are combined by means of the respective lines 16 and 18 in a mixing station 20. The combined sample portion and the particles 14 are mixed, and then allowed to settle differential by means of gravity sedimentation as shown by a block 22. Sample 12 and particles 14 are mixed to facilitate rapid binding of the particles to the selected cells of interest. The mixture of the sample 12 and the particles 14 is made to cause the particles 14 to make rapid contact with the selected cells of the sample 12. One advantage of the dense particles 14 is that they will settle by gravity differentially through the sample 12. followed by mixing without substantial trapping of non-selected or non-objective cells. During mixing, another advantage of the particles 14 is that the mixing is performed to cause the particles 14 to repeatedly pass or settle repeatedly through the sample to provide a cell-particle bond without physically damaging the cells with the particles. 14. For small volumes, in the order of microliters, the mixture can be rapid, such as vortex formation, as described in U.S. Patent No. 5,238,812, which is incorporated herein by reference. For large volumes, in the order of 0.5 milliliters to liters, an effective mixing method is to stir the sheets 14 and the sample 12 in an end-to-end manner. Once the particles 14 have been mixed with the sample 12, the particles 14 are allowed to settle to the bottom of a container (not shown), and then the sample fluid and the remaining cells can be separated as illustrated by a block 24. The particles 14 have a density sufficiently greater than the populations of the sample 12, both objective and non-objective, so that the particles 14 and the objective populations linked to them settle in a differential manner through the sample 12, leaving the unlinked / non-objective populations in suspension. For example, if the sample 12 is a blood sample, the blood cells have a density of the order of 1.05 grams / cubic centimeter, and therefore, the particles 14 must be substantially more dense than the cells, at least of the order of two. (2) to three (3) times denser than the cells. The sample fluid and the remaining cells can be removed for study, where the selected cells of interest have remained in the fluid and have been increased and do not bind to the particles 14. The particles 14 and the linked cells, they can also be removed from the remaining sample fluid, to remove the cells from the particles 14, if desired, for the study of the linked cells, where these are the cells of "interest." The fluid and the remaining cells are also they can be reinserted into a living organism, without the particles and cells bound thereto, which it is desired to remove from the sample or fluid.The apparatus 10 can be an automatic device that combines the sample 12 and the particles 14, and moves them between stations, or it may be a manual procedure, such as that performed by an operator using a test tube or a container for stations, 20, 22 and 24, or it may be a combination of the two procedures. the settling and separating steps 22 and 24 can preferably be performed by gravity separation only, additional steps can be included where desired, sample 12 and particles 14 can be n centrifugate briefly, as illustrated by a block 26, to accelerate the settling step 22. The particles 14 can also be made of a magnetic material. With the magnetic particles 14, a magnet or a magnetic field, illustrated by a block 28, can be applied to the bottom of the container (not shown) to accelerate the settling step 22. Additionally, the magnetic field 28 can be maintained or can be maintained. Apply to the bottom of the container to ensure that the particles 14 are not removed in the separation step 24. The remaining sample can be removed and passed through or through a magnetic field 30, to ensure that no fragments of particles or particles remain 14 in the fluid sample, such as when the sample is to be reinserted into a living organism, such as the human body. Referring now to Figure 2, a conceptual diagram illustrates a particle 14 having two different antibodies A and B bonded thereto. For exemplary purposes, a pair of A 32 positive cells that include at least one A 'antigen, which will specifically bind to an antibody bound A on the particle 14, are illustrated. A pair of B 34 positive cells are also illustrated which include minus a B 'antigen, which will bind specifically with a bound antibody B on the particle 14. In reality, there would be no particular order for the binding of the cells, and generally there would be a positive cell A or B blocking the particle view 14 on both free sides of the particle 14 (not shown). Also, antibodies A and B on a particle 14 bind to a single cell expressing antigens A '- and BA For example, if cell A was a positive CD4 cell, and cell B was a positive CD8 cell, then there would be four or five A cells, and only one or two B cells bound to particle 14. Although two different antibodies A and B are described as both bound to particle 14, each antibody can bind to separate particles 14 as seen. Again, as previously reported, the target or selected cells can be removed from the sample 12 bound to the particles 14, and then the cells can be removed from the particles 14 for a separate study of the cells. The cells can be removed from the particles 14 by conventional technology, such as biochemical separation or mechanical alteration methods. Although the specific particle 14 is not critical, a high density magnetic particle 14 is preferable. A preferable particle 14 is formed of nickel carbonyl, such as the nickel powders made by INCO as Nickel Powder Type 123. The particles 14 of preference is made with a nominal diameter of approximately five (5 microns), with a preferable scale of three (3) to thirty-five (35) microns, but not limited to it. Fine portions (smaller fragments) are removed before use. The particles 14 are relatively heavy, with a density of preference of the order of nine (9) grams / cubic centimeter. The density of the particles is selected such that the particles settle differentially through the sample suspension more rapidly than the cells. Accordingly, the objective cells bound to the particles will be separated by gravity before any significant isolation by settling of the cells or linked (non-objective). Clearly, the greater the differences in density between the sample populations and the particles 14, the faster the differential settlement will occur. The volumes of the sample fluid vary, depending on the procedure being carried out. For the analysis of blood, bone marrow, or spinal fluids, as few as ten (10) microliters may be used, although for clinical transplants, such as bone marrow, volumes may be approximately one hundred (100) milliliters to three (3 liters. Bone marrow procedures are typically purging procedures to remove unwanted cells from the bone marrow fluid. In whole blood or in the bone marrow, many procedures can be used, such as isolation of embryonic stem cells by removing the other blood cells and linking them with one or more monoclonal antibodies linked to one or more of the sets of particles 14. A preferred method for mixing the particles 14 with the sample 12 is to lightly stir the particles 14 and the sample mixture end to end, causing the particles 14 to fall repeatedly through the sample 12 to bind to the population of interest. This seems preferable, but the stronger oscillating or familiar mixing procedures can also be effective, if physical damage to the cells of interest is avoided by the dense and heavy particles 14. One of these devices can be a fastener Test tube that rotates slowly to rotate the test tube or similar end-on-end vessel. This allows having a "light mixture" of the particles 14 and sample 12 where the particles 14 mix and settle through a substantial portion of the sample in each rotation, allowing the target cells to bind with the particles, without apparent physical damage to the cells. The same mixing movement can be obtained by rotating or oscillating the tube back and forth, each end being first on the top and then on the bottom, in a manner similar to end-to-end rotation. The. The oscillator roller speed can also be set to perform substantially the same mixing procedure. An embodiment of an end-to-end blood bag mixer is illustrated in Figures 3A-3E and is generally designated by the reference numeral 40. A blood bag (not shown) is inserted into the mixer 40 by the release of a portion of the upper fastener 42 and lower 44. The upper 42 and lower portions 44 include an instant closure 46 that holds the portions joined together. The upper and lower portions 42, 44 are also preferably articulated to each other by a hinge 48. Although illustrated in a horizontal position in Figures 3A and 3B, the mixer 40 would be oriented in a substantially vertical manner to provide the extreme desirable mixture. on end of the sample fluid 12 and particles 14. The mixer 40 can be mounted on a substantially horizontal motor shaft shaft (not shown) by a fastener 50 (Figure 3E). The fastener 50 is attached to a portion 44 of the mixer 40. The fastener 50 can be attached to the portion 44 through a plurality of openings 52 (Figure 3D) formed in the portion 44. The fastener 50 includes a plurality of passages. coupling 54 (Figure 3E), which can be aligned with the openings 52, and then the fastener 50 can be mounted and secured by a plurality of screws or other fasteners (not shown). The fastener 50 is mounted by a passage 56 to an arrow of the motor (not shown) to rotate therewith through one or more threaded passages 58.

A screw (not shown) can be threadedly inserted into the passage 58 to be supported by the arrow of the motor to rotate the mixer 40. Like the blood tube, the blood bag in the mixer 40 is slowly rotated, and makes the particles they mix and settle through a substantial portion of the sample in each rotation to bond with the sample cells of the sample. A preferred method for labeling particles with antibody, which is effective to deplete specific cell subpopulations of a sample mixture, i.e., whole blood, bone marrow, mixed cell populations, or body fluids, and to the density of the particles, which settle easily in a differential way by the force of gravity, thus eliminating specifically bound cells together with the particles. Materials: Nickel particles - obtained from Novament Specialty Products Corp. (Yckoff, NJ 07481), a batch of Type 123 Nickel Powder from INCO (Sufferix, NY 10901). This batch had been screened through a 400 mesh to remove large particles, and classified as coarse. The resulting lot was calibrated in Fisher at 5.7 microns, with a surface area of 0.34 square meters / gram of particles. Regulator A - Tris / NaCl, pH 7.2 9.55 grams / liter of Tris 4.0 grams (liter of NaCl are combined in dH20, brought to a pH of 7.2 with concentrated HCl Regulator B - Tris / NaCl / BSA is added to Regulator A , Iovino Whey Albumin (ASB), 0.2 grams / 100 milliliters Antibody Solution - The amount of antibody that will need to be added to the particles is determined from the Table 1 for the particle labeling. the antibody material solution that will result in the required amount of purified antibody required.The amount of the material solution is measured and added to Regulator A just before the addition to the particles. nickel (1 gram of particles / 0.34 square meters of surface area is calculated) 2. The particles are washed twice with dH20.2a dH20 is added to the particles, and mixed by swirling and inverting the tube . 2b The particles are separated from the fluid by allowing them to settle for approximately 2 minutes and removing the fluid. 3. Wash the particles as in step 2, using a 1 percent bleach solution. 4. The particles are washed as in step 2, with Regulator A. 5. The nickel particles are optimally masked with bovine serum albumin. 5a. The particles are resuspended to a volume of 2 milliliters / gram of particles in Regulator A. 5b. An amount of 50 milligrams / milliliter of bovine serum albumin solution is added (1: 4 dilution of Regulator B) to result in a final concentration of 3 milligrams of bovine serum albumin / square meter of particle surface. 5c. The tube is placed on a roller for 3-6 hours at room temperature. 5 d. The particles are washed twice, as in step 2, with Regulator A, with 30-60 minutes on the roller between washes. 6. Addition of antibody to the particles. 6a. The granules are resuspended 'masked', Step 5, up to 1 milliliter / gram of granules. 6b The granules are sonified while the previously prepared antibody solution is added. 6c. The tube is rinsed where the Antibody Solution with Regulator A was made, and this is combined with the particles. 6d. The volume is brought to 1 milliliter / gram with Regulator A. 7. The tube is placed on a roller, and the particle / antibody solution is rolled overnight at ambient temperature. 8. The particles labeled with bovine serum albumin are blocked. 8a. The particles are washed twice with Regulator A, as in step 5d. 8b. HE add a volume of Regulator B in 2 milliliters / gram to the particles. 8c. The particles are resuspended by sonification and vortex formation. 8d. The particles are rolled for 1 hour. 8e. Regulator B is replaced twice with bearing for 30 minutes between exchanges. 9. Storage of labeled granules. 9a. The final blocking regulator B is removed. 9b. Fresh Regulator B is added up to a volume of 2 milliliters / gram. 9c. Antibody coated granules are stored in a capped tube at room temperature. 10. Test of antibody / particle preparation. 10a. Graduated amounts of antibody / particles are added to the test tubes, normally in the range of 10-200 microliters of antibodies / particles per milliliter of whole blood. 10b. The antibodies / particles are washed three times with three times the volume of a mixture of Isoton III (Coulter / Diagnostics) and Glucose (4.5 grams / liter). 10c. The IG solution is decanted and 1 milliliter of whole blood (collected either in EDTA or in Heparin anticoagulant) is added. lOd. It is mixed by extreme turning over end for 4 to 5 minutes. lOe. The tubes are placed vertically in a test tube holder for 4 to 5 minutes. lOf. The blood above the particles is transferred with a pipette to another tube (the pipette can be held against a magnet during this transfer to remove the nickel particles that may not settle during step 10). log. They are analyzed, using the best method to / To the exhausted population, and the residual cells are compared with the original population present in the original sample. lOh. An amount of particles that will be required per milliliter of whole blood is selected to effectively eliminate the population of cells in question. The invention is particularly adapted to bind microspheres with platelets and populations of white blood cells or populations of subsets of white blood cells. As used herein, Populations of subsets of white blood cells are subsets of a population of white blood cells with which specific monoclonal antibodies can be linked. The World Health Organization and the International Society of Immunology have defined a nomenclature for monoclonal antibodies. Monoclonal antibodies are defined by a naming agglomeration designation (DA) that defines a particular specificity for a cell or group of cells and monoclonal antibodies specific for that agglomeration designation group. For example purposes only, the agglomeration designation groups have been specified in the following table, together with the Coulter antibody designator. TABLE I ANTIBODY AMOUNTS FOR THE PREPARATION OF AND. NICKEL PARTICLES FOR EXTRACTION Particle Labels An t i c u erpo / D e ssu re (Amount / M2) • Platelet CD41 PLT-1 (3 mg) Cell B CD20 Bl (9 mg) CD19 B4 (3 mg) MY CD14 MY4A (10.5 mg) CD33 MY 9 (4.5 mg) MT4 CD2 Til (6.75 mg) CD5 TI (4.6 MG) CD7 3A1 (2.25 mg) CD26 TAI (1.5 mg) T4 CD4 T4 (5 mg) T8 CD8 T8 (5 mg) KC-48 CD15 KC-48 (5 g) HLA-DR 13 (5 mg) EXAMPLE 1 PREPARATION OF POPULATION OF GRANULOCITS TO START WHOLE BLOOD A portion of a whole blood sample collected in EDTA was tested on a Coulter STKS instrument (which removes red blood cells by lysis) as the control for the following depletions, as illustrated in Figure 4A. The control illustrates normal population patterns of white blood cells using DC (Coulter volume) and light scattering (DL) parameters, including populations of Lymphocytes 60, Monocytes 62, granulocytes (N 64 and Eosinophils 66) and a portion of waste 68. The appropriate amount of nickel particles coated with antibody and previously titrated was placed in a 12 x 75 millimeter glass test tube and washed three times by gravity settling with a solution of Isoton II (Coulter Corporation) containing 4.5 grams / liter of glucose (IG regulator). 3 milliliters of the ejitera blood sample were added to the washed particles, and the tube was capped. The tube was then placed on an end-to-end roller at approximately 30 revolutions per minute. It was found that this is appropriate to keep the particles in suspension, allowing the particles to fall repeatedly through the blood. Blood and particles were mixed on this roller for 4 minutes. Following the mixing, the tubes were removed and set up vertically in a rack of test tubes for 4 minutes to provide differential gravity settling. To analyze the remaining populations, the blood that was above the particles was transferred by means of a pipette to another tube. In some cases, the barrel of the pipette was held against a magnet to ensure removal of fine portions of nickel that may not have settled. The samples were then tested on a Coulter STKS instrument, and compared against the whole blood control. A description of this operation is disclosed in U.S. Patent No. 5,125,737, which is incorporated herein by reference. Although the particles - and whole blood are mixed, and the particles are bound to the white blood cell population or to the population of the subset of white blood cells, the red blood cells in each case are removed by lysis before obtaining the illustrated results . The results of platelets and red blood cells referred to herein are obtained using only one volume parameter of Coulter (DC) in instrument channels separated from the channel of white blood cells. The whole blood sample portion was depleted using the following labeled nickel particles: Y4 200 microliters / milliliter, T4 100 microliters / milliliter, Bl 100 microliters / milliliter, PLT-1 80 microliters / milliliter, T8 50 microliters / milliliter, and 3A1 50 microliters / milliliter. This mixture of labeled particles bound with antibody depleted most of Monocytes 62 ', 60' Lymphocytes, and platelets, to give an enriched population of granulocytes (N 64 'and Eosinophils 66') as illustrated in Figure 4B. Exhaustion resulted in an 86 percent reduction of platelets, and a clearing of the populations of Monocytes 62 '' and Lymphocytes 60 'in the exhausted sample, compared to whole blood (Figure 4A). This resulted in a cell preparation consisting of 98 percent of 64 'and 66' granulocytes, which was 91 percent of the original granulocyte number. The red blood cells were retained in 91 percent of the control whole blood sample, indicating the specificity of cell depletion. EXAMPLE 2 PREPARATION OF LYMPHOCYTES USING NICKEL PARTICLES WITH DISTRIBUTION AND RETENTION OF LYMPHOCYTES A lymphocyte preparation was made using nickel particles coated with antibody, as illustrated in Figures 5A and 5B. The results of a whole blood control test in a Coulter STKS hematology analyzer are illustrated in Figure 5A. The normal pattern yielded 27 percent of Lymphocytes 70, 9 percent of Monocytes 72, 61 percent of Neutrophils 74, and 2 percent of Neutrophils 74, and 2 percent of Eosinophils 76. One combination of the appropriate amount of nickel particles previously labeled with PLT-1 (70 microliters of particles / milliliter of blood), KC-48 (50 microliters of particles / milliliter of blood), and MY-4 (100 microliters of particles / milliliter of blood) for 10 milliliters of whole blood, was placed in a polystyrene test tube, and washed three times with IG regulator. The supernatant from the last wash was removed, and 10 milliliters of whole blood that had been collected in EDTA were added to the particles. The particles were resuspended in the blood, and placed on an end-to-end screw for 4 minutes. Following the mixing, the tube was placed vertically in a grid of test tubes, and left for 4 minutes to allow the particles to settle differential. After the particles had settled, the spent blood was removed with a plastic transfer pipette, and placed in a new tube. During the transfer, the barrel of the transfer pipette was held against a magnet, which removed any fine portions of nickel. The analysis of the lymphocyte preparation in a Coulter STKS hematology analyzer (Figure 5B) showed that the lymphocyte population improved up to 92 percent. The reduction of the other cell populations, compared to whole blood (Figure 5A) showed a platelet reduction of 94 percent, Neutrophils 74 'of 98 percent, Monocytes 72' of 80 percent, Eosinophils 76 '95 percent, and 1 percent red blood cells. This showed that the resulting exhausted sample consisted of 70 'lymphocytes with very little non-specific elimination (retention of more than 99 percent of the lymphocytes). EXAMPLE 3 RECOVERY OF T8 CELLS FROM NICKEL PARTICLES LABELED WITH ANTIBODY AFTER THE DEPLETION OF WHOLE BLOOD Initially, nickel particles labeled with T8 antibody were used to deplete the whole blood of T8 lymphocytes. Nickel particles labeled with T8 at a suboptimal dose for depletion (15 microliters against 50 microliters / milliliter of whole blood) were washed three times in IG regulator. Whole blood was added to the particles, and they were placed on an end roller on end for 10 minutes, and they were set vertically for 5 minutes. Exhausted blood was eliminated, and the bound particles and cells were washed twice by resuspension in a volume equal to half of the original amount of sample with IG regulator, inverting slightly, the particles and bound cells were allowed to settle. Following washing, the particle / cell granule was resuspended in the IG regulator, and placed on a magnetic stirrer for mechanical alteration for approximately 30 seconds. The alteration results in the separation of the cells from the particles. The supernatant was separated from the particles allowing the particles to settle, removed by pipette, and analyzed by flow cytometry. Three samples were analyzed; a control of whole blood, blood followed by depletion with the T8-labeled particles, and the supernatant with the cells released after the particles / cells had been agitated and the particles were allowed to settle. The results of Profile II demonstrate the normal appearance of depleted blood with a slight reduction of 16 percent of the lymphocyte population. However, the recovered cells demonstrate a highly enriched and purified lymphocyte population. Following analysis with fluorescent surface markers for T4 and T8, the depleted blood had the T8 population reduced from 26 percent to 6.3 percent; however, the T4 population increased from 52.5 percent to 66.1 percent, due to the decrease in T8 cells. In the recovered population, more than 96 percent of the cells were T8 positive.

EXAMPLE 4 LABELING OF DIFFERENT TYPES OF DENSIVE PARTICLES STUDY A: Different types of dense particles mentioned in Table II were labeled by the present method with T8 antibody. The normal procedure was used for the labeling of Nickel Type 123 particles for the different types of particles, which included blocking with 3-30 milligrams / square meter of bovine serum albumin, and labeling with 5 milligrams / square meter of T8 antibody . Following labeling and washing, T8 depletion of whole blood was performed in the normal method with titration of the amount of tagged particles added. Following a 4 minute mixture of the blood and the particles, the particles were allowed to settle for 4 minutes. The resulting depleted samples were then analyzed on a flow cytometer (Coulter II Profile) by the percentage of T8 cells. Depletion was calculated as the percentage of remaining T8 cells compared to the T8 value in whole blood. Nickel Type 123 is the particle used for the other experiments, and was the comparator for the other types of particles. From the titration, 25 microliters of Type 123 particles per milliliter of whole blood resulted in depletion of more than 96 percent of the T8 cells. A particle of stainless steel did not exhaust, even in 100 microliters of particles / milliliter of whole blood. The zinc powder, labeled with T8 antibody, resulted in the coagulation of the whole blood, probably due to its interaction with the EDTA anticoagulant, and because it causes activation of fibrinogen by releasing the calcium in the sample. Other types of nickel particles resulted in depletion, but not as effective as Type 123. STUDY B: Several different particle types were tested, labeled with T4 antibody, but without using a precoating step with bovine serum albumin. , for labeling by determining its ability to bind to cells. All the particles were bound with the antibody, as determined by this method. The Pb and the VM63-NÍ- were equivalent or slightly better to bind with the cells than the Type 123 -. 123 -Ni, but they settled slowly. The Ti02, the Pb, and the A ~ VM63-NÍ were all effective in labeling cells for microscopic identification. It was only shown that Ta is ineffective in binding to cells after being labeled with the antibody. STUDY C: The particles were labeled with antibody KC-48, specific for neutrophils, during the normal procedures for nickel type 123 particles. Then the particles were mixed with whole blood, a blood spot was made, and it was marked and examined using a microscope. All these particles demonstrated a specific bond with neutrophils. In summary, almost all the metal particles tested provided at least some degree of antibody adsorption. However, in the context of depletion capacity, Type 123 Nickel was more convenient due to its surface properties and settling speeds. As an example, the palladium and manganese dioxide particles also deplete, but failed to settle fast enough to be effective in the present invention. The antibody adsorbed on titanium dioxide particles provided efficient labeling of the cells for microscopic identification, but, due to the small size, did not result in a significant differential settlement in the whole blood. TABLE II r * - MATERIAL DESIGNATION MANUFACTURER / - CHARACTERISTICS CAPACITA CATALOG / LOT NO. PHYSICAL AND MAG. OF AGOT. Study A • 0 NICKEL TYPE 123 Novamet / 3451313 IRREGULAR NICKEL VM 63 Nóvame / VM63 ULTRAFILE OR NICKEL DUST 10 / 585A Novamet / I0 / 585A NICKEL SPHERES HDNP Novamet / 347355 STEEL POWDER P316L Ametek / 0813290 IRREGULAR FORM 5 INOX. Aldrich POWDER / HY13401CY NON MAGNETIC ZINC Study B: NICKEL TYPE 123 Novamet / 3451313 NICKEL VM 63 Novamet / VM63 ULTRAFINE POWDER NICKEL 8 / 209A Novamet / 8 / 209A NICKEL SPHERES 08841R Spex Ind./08841R NICKEL POWDER 01509 BW Aldrich / 01509BW NICKEL DUST 347355 Novamet / 347355 Pd D13A17 POWDER John Matthey Elec./- ++ D13A17 TiO. ANATASA NON MAGNETIC Ta SGQ Norton Metals Div./- SGQ-2-3764 SiO "NO MAGNETIC NiO, NO4990 Pflatz & Bauer / 040291 NOT MAGNETIC Study C: Pd D13A17 John Matthey Elec./- Approx.0.5 μm diameter D13A17 UNCLE, ANATASE Approx .0.5 μm diameter. MnO. Aldrich / 23, 094-4 POW SGQ Norton Metals Div./- POWDER SGQ-2 -3764 * The zinc added to whole blood resulted in coagulation.

EXAMPLE 5 EXHAUSTION OF SUBSTITUTE T4 AND T8 OF WHOLE BLOOD USING NICKEL PARTICLES LABELED WITH ANTIBODY Nickel particles were labeled with either T4 or T8 antibody, using the procedure previously referenced for antibody labeling. For depletion, the particles (50 microliters / milliliter of whole blood) were transferred to a test tube, and washed three times with IG regulator. Following the removal of the third wash, whole blood was added to the particles, and the combination was mixed, in an end-to-end manner, for 4 minutes. Following the mixing, the tubes were placed in an upright position, and the particles were allowed to settle for 4 minutes. The spent blood was then labeled with fluorescent antibody T4-RD1 / T8-FITC (Coulter Corporation, Coulter Cytostat, part no.6603802), and assayed on a flow cytometer (Coulter Profile II). All samples were counted for 1 minute, and populations of different quadrants were compared by T4 and T8 lymphocytes. Compared to whole blood control, when T4 particles were used, 94 percent of the T4 population was depleted, while only 18 percent of T8 was removed. When T8 particles were used, 96 percent of the T8 population was depleted, while only 4 percent of the T4 population was eliminated. EXAMPLE 6 DIFFERENTIAL SETTING FIGS. 6A-C illustrate the results of the differential settlement of the dense particles of the present invention, contrasting with the magnetic particles Rhone-Poulenc of the prior art. Figure 6A again illustrates a control histogram in a STKS, which includes a > - Normal population pattern of Lymphocytes 80, Monocytes 82, Neutrophils 84, and Eosinophils 86. Figure 6B illustrates the pattern resulting from a depletion of nickel particles, using particles with a monoclonal antibody label KC48. The neutrophils 84 were 59.6 percent of the results of the blood white cell control population illustrated in Figure 6A, while the 84 'neutrophils were reduced to 2.3 percent of the white blood cell populations illustrated in Figure 6B. Rhone-Poulenc particles were used in a manner similar to the nickel particles, and virtually show no settlement by gravity, as illustrated by the histogram of Figure 6C. In particular, bound neutrophils and Rhone-Poulenc particles show a pattern 88, while unbound Rhone-Poulenc particles appear as a noise or waste pattern 90. The Rhone-Poulenc publications assert "that without a magnetic field, no significant sedimentation takes place for several hours ", again indicating that these particles are designed to prevent settlement by gravity. EXAMPLE 7 MIXING TIMES The mixing times and methods can be varied according to the sample volume and the desired incubation times. For volumes in the order of 0.5 milliliters or less, both rapid mixing such as swirling or nutation and end-to-end settling of the dense particles can be effectively used without physical damage to the cell populations. Swirl formation was performed using antibody-bound particles separated from KC48-Nickel (50 microliters / milliliter of whole blood) and PLT-Nickel (100 microliters / milliliter of whole blood) with the results of the Coulter STKS illustrated in Table III. In Table III, and in each of the other similar tables, such as Tables V, VIII, IX, X, and XII, platelets and white blood cells are summarized in units of 103 / microliter, while red blood cells are in units of l06 / microliter.

TABLE III GBS N L M PLT GRS A) 5.6 2.8 2.1 0.5 230 4.09 B) 4.1 1.4 2.1 0.4 91 4.19 C) 3.4 0.7 2.0 0.5 58 4.15 Example A was a vortex control for 20 seconds without particles, - Example B included the swirl particles for fifteen (15) seconds, and Example C included the swirl particles for thirty (30) seconds and seated in each case for four (4) minutes. In conclusion, neutrophils were depleted in fifty (50) percent, and platelets were depleted in sixty (60) percent, over a swirl formation of fifteen (15) seconds, while fifteen (15) additional seconds they increased neutrophil depletion up to seventy-five (75) percent, and platelets up to seventy-five (75) percent. It was also noted that the other cell populations were retained without non-specific losses. The same blood sample was mixed end to end for different times, as illustrated in Table IV.

TABLE IV A) Control, without particles, 10 minutes B) KC48 / PLT, 30-45 seconds. C) "", 1 minute. D) "", 1.5 minutes. E) "", 2.0 minutes. F) "", 4.0 minutes. G) "", 10 minutes.

The depletion results obtained by the mixing procedure of Table IV are illustrated in Table V. When the STKS instrument reports a result of 0.0 (Neutrophiles in Table V, F or G), the actual result is below 0.05. , in general more than 99 percent. TABLE V (% DEPLETION) Results: GBS N L M PLT GRS Control A) 5.6 2.8 2.1 0.5 240 4.20 . 45 sec. B) 3.7 1.0 (64%) 2.0 0.5 69 (71%) 4.08 1 min. C) 3.2 0.6 (79%) 1.9 0.5 39 (64%) 4.02 1. 5 min D) 2.8 0.4 (86%) 1.8 0.4 37 (85%) 4.00 2. 0 min. E) 2.9 0.3 / 89%) 1.9 0.5 14 (94%) 4.10 4. 0 MIN. F) 2.6 0.0 (> 99%) 2.0 0.5 1 (99.6%) 4.19 minutes. G) 2.7 0.0 (> 99%) 2.0 0.6 0 (> 99.6%) 4.22 For these particles and antibodies, the minimum mixing time appears to be approximately four (4) minutes. For other particles and antibodies, the mixing time may vary within the scope of the present invention. Clearly, a minimum mixture may be desirable beyond the minimum time in some cases, and is not detrimental to the present invention. EXAMPLE 8 SMALL SAMPLE VOLUMES Small volumes of 20 microliters of whole blood were numbered with 5 microliters of KC48 nickel particles for four (4) minutes. The results showing the elimination of 95 percent of the neutrophils, were obtained from a conventional whole blood spot assay, as shown in Table VI. TABLE VI N _____ _M_ _E_ Control, without particles 58 27 12 2 Exhausted 3 82 11 3 A second small volume of 10 microliters of whole blood was fed with 1 microliter of KC48 and 2 microliters of PLT-1 for four (4) minutes. The results were an 82 percent depletion of granulocytes obtained in a Profile II flow cytometer.

EXAMPLE 9 ELIMINATION OF GRANULOCYTES AND / OR PLATELETS OF SAMPLE PREPARATIONS Platelets are a component of whole blood and bone marrow which, during the preparation of cell suspensions, are removed by different methods. The attributes of the platelets that make them effective in the repair of wounds, are inconvenient in the work of cellular preparation, that is, the coagulation of platelets and non-specific adhesion to other cells. Since there are approximately 20 to 50 platelets per leukocyte in whole blood, removal of the platelets before any separation work increases the recoverability of the leukocytes, resulting in a leukocyte profile that more closely resembles that of the whole blood, and decreases the time of preparation, since the most common method to remove platelets is by three separate centrifugations at low speed after the cell suspension is isolated. In a preparation that is to be administered to a patient, the elimination of platelets before freezing would decrease the non-specific loss of the cells to be infused, and eliminate platelet aggregates. In addition, mature granulocytes contain granules that, upon release, can result in a shock to a patient upon infusion. By eliminating both mature granulocytes and platelets, the cellular preparation for infusion, either immediately or immediately after freezing, would be safer and less problematic for the patient. Figure 7A illustrates a control whole blood population containing lymphocytes 100, Monocytes 102, Neutrophils 104, Eosinophils 106, and platelets (not shown). The control platelets were 276 x 10 3 platelets / microliter, while the granulocytes (neutrophils and Eosinophils) were 3.2 x 10 3 / microliter. Two sets of dense particles were combined and mixed with the blood for 4 minutes, and settled for 4 minutes. One set of particles included particles labeled with PLT-1 at 80 microliters / milliliter, and the second set of particles included particles labeled with KC48 at 50 microliters / milliliter. As illustrated in Figure 7B, 100 'lymphocytes and Monocytes 102' were affected very little, while Neutrophils 104 'and Eosinophils 106' were reduced by approximately 99.9 percent. The platelets were reduced to approximately 2 x 10 3 / microliter. Platelets and granulocytes can also be removed separately in separate blood sample portions. Figure 8A illustrates a control whole blood population containing Lymphocytes 110, Monocytes 112, Neutrophils 114, Eosinophils 116, and platelets (not shown). Figure 8B illustrates a sample portion following platelet depletion, again using the dense particles labeled with PLT-1. Platelets were reduced from 231 x 103 platelets / microliter in the control whole blood population to 3 x 103 platelets / microliter in the exhausted sample portion. The remaining populations of Lymphocytes 110 ', Monocytes 112', Neutrophils 114 ', and Eosinophils 116' were relatively unaffected. Figure 8C illustrates a sample portion following the depletion of Neutrophils 114 and Eosinophils 116, using dense particles labeled with KC48. Neutrophils 114"and Eosinophils 116" were reduced to essentially zero from a total of Neutrophils 114 and Eosinophils 116 of 2.8 x 103 / microliter. Platelets were relatively unaffected. EXAMPLE 10 INCREMENTED GRAVITY SETTING Figures 9A-9F illustrate gravity settling histograms as compared to a brief accelerated settling using the particles of the present invention. Figures 9A-9D illustrate a Neutrophil preparation that uses the particles with the labels mentioned in Table VII.

TABLE VII T4 50 μl / ml T8 50 μl / ml MY4 50 μl / ml BL 50 μl / ml PLT 40 70 μl / ml 13 50 μl / ml Figure 9A again illustrates a population of control whole blood of Lymphocytes 120, Monocytes 122, Neutrophils 124, Eosinophils 126, platelets (not shown), and red blood cells (not shown). TABLE VIII FIG. SAMPLE GBS N L M E PLT GRS 9A Control 6.9 4.1 2.2 0.4 0.2 288 4.87 9B dep. Settlement 4.3 3.9 0.2 0.0 0.1 35 4.59 9C centr. Control 6.9 4.1 2.2 0.4 0.1 297 4.74 9D dep. Centrifugal 4.3 3.9 0.2 0.0 0.1 15 4.60 9E centr. Control 7.4 4.5 2.2 0.4 0.2 293 4.96 9F dep. Centrifugal 2.7 0.0 2.1 0.4 0.1 280 4.94 The preparation of Neutrophils using the particle tags of Table VII results in an enriched population of Neutrophils 124 ', where the percentage of neutrophils in the white blood cells has increased from 59.7 percent to 89.6 percent. Lymphocytes decreased from 32.1 percent to 4.9 percent, and Monocytes decreased from 5.5 percent to 0.8 percent, as illustrated in Figure 9B. Figure 9C illustrates a population of whole blood Lymphocytes control 130, Monocytes 132 134 Neutrophils, eosinophils and 136. In this example, instead of gravity settling the sample portion and the particles were centrifuged on a small centrifuge, such as a Fisher Scientific Model 59A Microcentrifuge, for 15 seconds in the No. 2 position. Short centrifugation or increased / enhanced gravity settlement obtained similar results to gravity settling in a shorter period of time, if desired. The percentage of neutrophils increased from 59.5 percent to 89.1 percent. The lymphocytes decreased from 31.5 percent to 5.7 percent, while the Monocytes decreased from 5.8 percent to 0.5 percent, as illustrated in Figure 9D. Any population or separate subpopulation can be eliminated using the same procedure, for example, as illustrated in Figures 9E and 9F. Figure 9E illustrates the population of whole blood Lymphocytes control 140 142 Monocytes, Neutrophils 144, and 146. In this example Eosinophils, Neutrophils 144 are removed using enhanced gravity centrifugation of the sample and particles. Neutrophils are reduced from 61.4 percent in the control, to 0.7 percent, as illustrated by 144 'in Figure 9F, while the remaining populations are relatively unaffected. The primary aspects of the present invention relate to the settlement by gravity of the dense particles. However, increased gravity settling with the cells could be used in a density gradient system, such as ficoll, in which case, only the particles would be required to be slightly more dense than the cells and the gradient system. With increased gravity settling (centrifugation), slightly denser particles and cells bound thereto could be separated in the ficoll gradient system. EXAMPLE 11 SETTING TIME Figures 10A-10F illustrate histograms comprising different results of the gravity settling time of the present invention, the results of which are summarized in Table IX.

TABLE IX FIG. SAMPLE GBS N L M GRS PLT 10A Control 4 min. 8.2 3.5 3.3 1.0 5.45 315 10B Exhausted 4 min. 5.0 0.1 3.4 1.1 5.44 315 10C Control 2 hours 8.4 3.7 3.2 1.0 5.08 350 10D Exhausted 2 hours 4.6 0.1 3.3 1.0 5.33 319 10E Control 3 hours 8.3 3.6 3.2 1.0 5.48 321 10F Exhausted 3 hours 4.6 0.0 3.4 1.0 5.31 321 The results of Table IX were obtained by adding 3 milliliter portions of the same whole blood population sample to four separate tubes or containers. The first tube was a control tube, and to each of the other three tubes were added 120 microliters of particles labeled with KC48. Then the four tubes were mixed end to end for four (4) minutes, and then they were allowed to settle by gravity during the respective times of four (4) minutes, doe (2) hours, and three (3) hours. The portion of samples that was above the particle was then stirred, mixed, and analyzed. Then a control portion (Figures 10A, 10C, and 10E) was compared to the respective exhausted samples (Figures 10B, 10D, and 10F). As shown by the figures and Table IX, the whole blood populations of the control portion remained virtually unchanged on the scale of four (4) minutes to three (3) hours. Also, as illustrated, the spent portions for each settling time are substantially the same. For the four (4) minute settling example, Neutrophils 150 (Figure 10A) were reduced from 42.9 percent to Neutrophils 150 '(Figure 10B) of 2.5 percent. In the same way, in the example of aeration of doe (2) horae, neutrophils 152 (Figure 10C) were reduced from 43.5 percent, to Neutrophils 152 '(Figure 10D) of 1.1 percent. In the example of settlement of three (3) hours, Neutrophils 154 (Figure 10E) were reduced from 42.7 percent to Neutrophils 154 '(Figure 10F) of 0.8 percent. EXAMPLE 12 PARTICLE HEATING FIGS. 11A-11C illustrate the comparison of Type 123 -Ni particles not heated with Type particles. 123 -. 123 -Ni heated, as also tabulated in Table X. TABLE X FIG. SAMPLE GBS N L M GRS PLT HA Control 6.4 3.9 1.8 0.5 4.28 337 11B Not heated 2.4 0.0 1.9 0.4 4.14 311 11C Heated 2.4 0.0 1.9 0.4 4.19 307 Again, the results were obtained by using particles labeled with KC48. The other populations were relatively unaffected, although the results of the unheated particles (Figure 11B) and of the heated particles (Figure 11C) were essentially the same. Before the adsorption of the antibody, the particles were heated at 250 ° C for three (3) hours, to be sterilized (to eliminate the microbes), and to eliminate the endotoxins from the particles, to be used especially where they were to be reintroduced. sample treated a patient. The heating of the particles also decreases the solubilization of nickel ions from the particles, by the formation of an oxide layer on the surface of the particles. The particles were allowed to settle for four (4) minutes after mixing for four (4) minutes as before. Neutrophils 156 (Figure HA) were reduced from 61.8 percent to Neutrophils 156 '(Figure 11B) of 1.4 percent, using unheated particles, and even neutrophils 156" (Figure 11C) of 1.7 percent, using the heated particles. In general, Type 123-Ni particles can be heated on a scale from 250 ° C to 280 ° C for three (3) to five (5) hours. Since the results of the heated and unheated particles were essentially equivalent, the other examples were not repeated, and reflect the use of the unheated particles.

EXAMPLE 13 IMPROVED CELLULAR PREPARATION FOR TRANSPLANTATION The particles of the present invention can also be used to deplete platelets in a bone marrow preparation (prep.). The conventional bone marrow processing methods were compared with the particle removal techniques of the present invention, as illustrated in Table XI. TABLE XI With eneiona1 Depletion of Particles / Platelets Percentage recovered after thawing. 29% 46% Viability 95% 99% Percentage of recovery of colony forming units. 56% 71% The conventional method of preparation of bone marrow uses separation on ficoll, followed by resuspended and washed progenitors harvested with three centrifuges at low speed to remove platelets.

^ The conventional technique example resulted in a recovery of 29 percent after thawing of the bone marrow, from where 95 percent were viable, and 56 percent of colony-forming units (CFU) were recovered ( or progeny cells). In contrast, the depletion of particles of the present invention, which is much faster and less complicated, resulted in a 46 percent recovery after thawing, with a 99% viability r1 and a unit recovery. colony formers 71 percent. Lae platelets were separated from the bone marrow with the particles before separation with ficoll. This eliminated conventional slow washings, reduced platelet / cell aggregates, which provided the best recovery of colony forming units. In the example illustrated in Table Xi, 30 milliliters of bone marrow were depleted using 600 microliters of the particles labeled with PLT-1., they were mixed end to end and settled, each one for four (4) minutes. The sample was then layered on ficoll, followed by thirty (30) minutes of centrifugation at 600 G. The interface was then harvested and concentrated by centrifugation in Tris / NaCl + 0.05 percent bovine serum albumin. The recovered cells were re-suspended in the RPMI 1640 + SBF (fetal calf serum) culture medium at 10 percent. Then the processed sample was frozen and thawed to be compared with the conventional methodology. As a further enrichment of the colony forming units, a small portion (1.3 milliliters) of the first sample of bone marrow depleted of platelets was further depleted, using particles labeled with 15 microliters of KC48 particles, 50 microliters of Til particles, 50 microliters. of particles labeled with Bl and B4, and 50 microliters of particles labeled with MY4 and MY9. This substantially eliminated all positive (mature) cells from the bone marrow line. Through depletion of mature cells, a highly enriched population of progenitor / embryonic stem cells (colony forming units) was recovered for analysis. The CFU-CM (granulocyte, monocyte) / 105 cells obtained in a sample before freezing using the conventional preparation, were 143 CFU-GM, which using the depletion of particle-platelets of the present invention, were 147 CFU-GM, while using the depletion of the additional particle line of the present invention, they were 620 CFU-GM. EXAMPLE 14 LYOPHILIZED PARTICLES As illustrated in Table XII, the lyophilized particles of the present invention were also effective in depleting neutrophils and platelets. Two sets of particles were combined, one labeled with KC48 and one labeled with PLT-1, to deplete neutrophils and platelets. TABLE XII SAMPLE GBS N L M PLT GRS Control of whole blood. 7.2 4.2 2.4 0.5 207 4.33 Whole blood with lyophilized particles. 3.1 0.1 2.5 0.4 6 4.45 In conclusion, lyophilized particles appear to be as effective as non-lyophilized particles. The lyophilized particles could be used in cases or in other uses, since freeze-dried particles eliminate the "**" requirement to keep the particles in solution. Many modifications and variations of the present invention are possible in light of the above teachings. Accordingly, it should be understood that within the scope of the appended claims, the invention may be practiced in a manner different from that specifically described.

Claims (35)

1. "" ~ NOVELTY OF THE INVENTION Having described the foregoing invention, it is considered as a novelty, and therefore, the content of the following is claimed as property: CLAIMS 1. A method for eliminating an e-selected eubpopulation of cells from a fluid sample of blood or bone marrow cells, said fluid sample comprising a plurality of different cell eubpoblatione comprising this method: providing a plurality of particles having a density at least twice the average cell density, and a size of at least 3 microns, said particles having attached thereto a reagent which binds with specific cells of the selected subpopulation; mixing these particles with a portion of the fluid mixture in a container to cause the particles to become bound only to the cells of the selected subpopulation, this mixing step being carried out causing the particles to settle repeatedly, due to the forces of gravity , through a substantial portion of the fluid sample; at the end of the mixing step, allow the particles and cells bound thereto to settle differentially under the forces of gravity, such that these particles become separated spatially with respect to the cells (not bound in said fluid sample, and separating at least a portion of the unlinked cells from the spatially separated particles and the cells linked thereto 2. The method according to claim 1, characterized in that the selected e-cell subpopulations are cancer cells, leukocytes, platelets, immune cells, or lineage-specific cells r 3. The method according to claim 1, characterized in that the mixing is effected by rotating the container to make the sample and the cells. particles are turned to extreme extreme 4. The method according to the claim in claim 1 , characterized in that the particles are formed of nickel. 5. The method according to claim 1, characterized in that the particles have a diameter of about 3 to 35 microns. 6. The method according to claim 5, characterized in that the particles have a diameter of about 5 microns. 7. The method according to claim 1, characterized in that the particles have a density greater than 2 grams / cubic centimeter. 8. The method according to claim 7, characterized in that the particles have a density of approximately 9 grams / cubic centimeter. 9. The method according to claim 7, characterized in that the particles have a density less than three times the deneity of the eanguineous cells of eubpopulation. 10. The method according to claim 1, characterized in that the reagent comprises: a drug, hormone, growth factor, lectin, enzyme, or nucleic acid sequence. 11. The method according to claim 1 wherein the reagent is an antibody. 12. The method according to claim 11, characterized in that the antibody is specific for platelets or for white blood cells. The method according to claim 1, characterized in that the mixing is carried out for approximately 15 seconds to 30 minutes. The method according to claim 13, characterized in that the mixing is carried out for approximately 4 minutes. 15. The method according to claim 1, characterized in that the differential settling is performed between approximately 15 eecond and 180 minutes. 16. The method of compliance with the claim in claim 15, characterized in that the differential settlement step is performed for approximately 4 minutes. The method according to claim 1, characterized in that the separation step includes removing at least a portion of the supernatant resulting from the sample portion without the particles and without the population or sub-population linked. 18. The method according to claim 1, characterized in that the separation step includes isolating these particles and subsequently eliminating the population or sub-population linked from said particles. 19. The method of compliance with the claim in the - *** _,. . . "Claim 18, characterized in that the linked subpopulations are CD8 positive cells. 20. The method according to claim 1, characterized in that the reagent is specific for eubpopulations of blood cells of monocytes, lymphocytes, and platelets, by which, the subpopulation of granulocytes becomes spatially separated from the subpopulations of monocytes. , lymphocytes, and platelets. 21. The method according to claim 1, characterized in that the specific reagent for the eubpopulations of blood cells of monocytes, neutrophils, eosinophils, and platelets, by which, the subpopulation of lymphocyte becomes separated from the subpopulations of monocyte, neutrophils, eoeinophilia, and platelets. 22. The method according to claim 1, characterized in that the reagent is specific for the eubpopulation of platelet blood cells, by which, the subpopulation of leukocyte cells becomes spatially separated from the platelet subpopulation. 23. The method according to claim 1, characterized in that the reagent is specific for the neutrophil subpopulation of blood cells. 24. The method according to claim 1, characterized in that the reagent is specific for substantially all lineage-positive cells. 25. The method according to claim 1, characterized by heating said particles before linking the reagent thereto. 26. The method according to claim 1, further characterized by lyophilizing said particles. 27. An apparatus for removing a previously selected subpopulation of blood cells from a whole blood sample, which comprises: a plurality of particles having a density at least twice the density of blood cells, and a diameter between about 3 and 35 microns, said particles having bound thereto a specific reagent for the previously selected cell subpopulation, the particles being adapted to be mixed with a portion of the whole blood sample to link said particles to the previously selected cell subpopulation, and making it possible for the particles and bound cells to settle differentially in the sample portion with respect to the unlinked cells. 28. The apparatus according to claim 1, wherein said particles comprise nickel. 29. The apparatus according to claim 27, characterized in that said particles have a density greater than 2 grams / cubic centimeter. 30. The apparatus according to claim 27, characterized in that said particles are made of nickel, and have a density of about 9 5 grams / cubic centimeter. 31. The apparatus according to claim 29, characterized in that said particles have a density less than three times the density of the eanguineous cells of that subpopulation. 32. The apparatus according to claim 27, characterized in that the reagent is at least one of an antibody, drug, hormone, growth factor, lectin, enzyme, or nucleic acid sequence. 33. The apparatus according to claim 27, characterized in that the reagent comprises an antibody specific for platelets or eanguineal white blood cells. 34. The apparatus according to claim 27, characterized in that the particles are lyophilized. 35. The apparatus according to claim 27, characterized in that these particles comprise nickel, and have a diameter of about 5 microns. 0