US3709791A

US3709791A - Method and apparatus for lymphocyte separation from blood

Info

Publication number: US3709791A
Application number: US00133524A
Authority: US
Inventors: B Lichtenstein
Original assignee: Technicon Instruments Corp
Current assignee: Bayer Corp
Priority date: 1971-04-13
Filing date: 1971-04-13
Publication date: 1973-01-09
Anticipated expiration: 1990-01-09
Also published as: NL170983B; DE2217266A1; CA955892A; CH544308A; DE2217266C2; GB1386303A; FR2132800A1; NL170983C; BE781532A; SE378309B; JPS5522445B1; IT968389B; FR2132800B1; NL7204570A; SU433695A3

Abstract

NEW AND IMPROVED METHOD AND APPARAYTUS FOR THE SEPARATION OF LYMPHOCYTES FROM WHOLE BOLLD SAMPLES UNDER STERILE CONDITIONS ARE PROVIDED AND COMPRISE STERILE, DISPOSABLE SYRINGE MEANS IN WHICH ARE MIXED LAND INCUBATED A BLOOD SAMPLE AND A SEPARATING AGENT. THE LATTER INCLUDES SENSITIZED MAGNETIC PARTICLES WHICH THE EFFECTIVE TO TAG THE BLOOD SAMPLE LEUKOCYTES TO THE SUBSTANTIAL EXCLUSION OF THE LYMPHOCYTES, AND AN ERYTHROCYTE SEDIMENTING AGENT WHICH IS EFFECTIVE TO PROMOTE THE SETTLING OF THE BLOOD SAMPLE ERYTHROYTES. FOLLOWING MIXTURE INCUBATION AND SETTLING IN THE SYRINGE, THE SAME IS OPERATIVELY POSITIONED ON SAMPLE TRANSFER AND MAGNETIC SEPARATOR MEANS WHICH INCLUDES A STERILE, DISPOSABLE SAMPLE TRANSFER TUBE AND AN OPERATIVELY ASSOCIATED MAGNETIC SEPARATOR. THE MIXTURE IS PUMPED AT SUBSTANTIALLY CONSTANT FLOW RATE FROM SAID SYRINGE, BY AUTOMATIC ACTUATION OF THE SYRINGE PLUNGER, FOR FLOW THROUGH SAID OPERATIVELY ASSOCIATED TRANSFER TUBE AND MAGNETIC SEPARATOR WITH RESULTANT SUBSTANTIAL RETENTION OF THE TAGGED LEUKOCYTES THEREIN AND CONTINUED FLOW OF THE LYMPHOCYTES IN THE PLASMA THROUGH THE REMAINDER OF THE TRANSFER TUBE TO LYMPHOCYTE COLLECTION MEANS.

Description

1973 B. LICHTENSTEIN 3,709,791

METHOD AND APPARATUS FOR LYMPHOCYTE SEPARATION FROM BLOOD Filed April 13, 1971 4 Sheets-Shoot 3 INVEN'IOR.

BERNARD LICHTENSTEIN Jan. 9, 1973 Filed April 13, 1971 METHOD AND APPARATUS FOR LYMPHOCYTE B. LICHTENSTEIN 3,709,791

SEPARATION FROM BLOOD 4 Sheets-Shoot 4 INVPINIOR. BERNARD LICHTENSTEIN ATTORNIQ United States Patent 3,709,791 METHOD AND APPARATUS FOR LYMPHOCYTE SEPARATION FROM. BLOOD Bernard Lichtenstein, Yorktown Heights, N.Y., assignor to Teelmicon Instruments Corporation, Tarrytown, N.Y. Filed Apr. 13, 1971, Ser. No. 133,524 Int. Cl. C12k 1/00 US. Cl. 195-127 6 Claims ABSTRACT OF THE DISCLOSURE New and improved method and apparatus for the separation of lymphocytes from whole blood samples under sterile conditions are provided and comprise sterile, disposable syringe means in which are mixed and incubated a blood sample and a separating agent. The latter includes sensitized magnetic particles which are effective to tag the blood sample leukocytes to the substantial exclusion of the lymphocytes, and an erythrocyte sedimenting agent which is effective to promote the settling of the blood sample erythroytes. Following mixture incubation and settling in the syringe, the same is operatively positioned on sample transfer and magnetic separator means which include a sterile, disposable sample transfer tube and an operatively associated magnetic separator. The mixture is pumped at substantially constant flow rate from said syringe, by automatic actuation of the syringe plunger, for flow through said operatively associated transfer tube and magnetic separator with resultant substantial retention of the tagged leukocytes therein and continued flow of the lymphocytes in the plasma through the remainder of the transfer tube to lymphocyte collection means.

BACKGROUND OF THE INVENTION (1) Field of the invention This invention relates to new and improved, semi-automated method and apparatus for the separation of lymphocytes from whole blood samples under absolutely sterile conditions and with 'very high combined degrees of purity yield and viability.

(2) Description of the prior art The separation of lymphocytes from whole blood samples under absolutely sterile conditions with very high combined degrees of purity, yield and viability is of particular advantage in the diagnosis of a very wide variety of serious human diseases as set forth, for example, in the copending application of myself and others also entitled New And Improved Method And Apparatus For Lymphocyte Separation From Blood Ser. No. 79,913, filed Oct. 12, 1970 and assigned to the assignee hereof.

Although a wide variety of methods and apparatus are known for the separation of lymphocytes from whole blood samples, it may be understood that no automated or semi-automated method and apparatus are known Which perform the lymphocyte separation under absolutely sterile conditions. Accordingly, the problem of inter sample contamination or the like, with attendant uncertainty as to the overall validity of the lymphocyte separation process, can arise. Too, it may be understood that many of the automated or semi-automated apparatus of the prior art are of relatively complex and costly construction to thus place the same beyond the realm of small laboratories or the like.

OBJECTS OF THE INVENTION It is, accordingly, an object of this invention to provide new and improved semi-automated method and ap- "ice paratus for the separation of lymphocytes from whole blood samples under absolutely sterile conditions.

Another object of this invention is the provision of apparatus as above which are of extremely simple and reliable construction to thus provide for a very low initial cost thereof and insure long periods of satisfactory, substantially maintenance-free operation.

A further object of this invention is the provision of method and apparatus as above which provide for lymphocyte separation with very high combined degrees of purity, viability and yield.

SUMMARY OF THE DISCLOSURE As disclosed herein, the method and apparatus of the in ention comprise the use of sterile, disposable syringe means to collect and mix measured amounts of a blood sample and a separating agent from respective containers thereof. Said separating agent comprises sensitized magnetic particles which, upon blood sample-separating agent mixture incubation effect the tagging of the blood sample leukocytes to the substantial exclusion of the blood sample lymphocytes, and a sedimenting agent to promote sedimentation of the blood sample erythrocytes. Following mixture and incubation of the loaded syringe in rotary mixer and incubator means, and standing of the syringe to enable erythrocyte sedimentation, the syringe is operatively positioned on sample transfer and magnetic separator means which include a sterile, disposable sample transfer tube sealed adjacent its respective extremities, magnetic separator means defining a generally circular path through which extends a high density, non-uniform magnetic field and through which said tube is passed, and a receptor tube assembly, respectively. In operation the syringe and receptor tube assembly are connected to opposite extremities of the transfer tube by hypodermic needle puncture of the latter, and the incubated blood sample separating agent mixture pumped by drive of the syringe plunger into and through the transfer tube and magnetic separator means with resultant retention of the tagged leukocytes in the latter and continued iiow of the lymphocytes in suspension in the blood plasma through said transfer tube into the receptor tube assembly.

DESCRIPTION OF THE DRAWINGS The above and other objects and advantages of the invention are believed made clear by the following detailed description thereof taken in conjunction with the accompanying drawings wherein:

FIG. 1 is a side elevational view of a disposable syringe utilized in the practice of the invention;

FIG. 2 is a cross sectional view of a separating agent container utilized in the practice of the invention;

FIG. 3 is a cross sectional view of a blood sample container utilized in the practice of the invention;

FIG. 4 is a front elevational view with parts in cross section of rotary mixer and incubator means constructed and operative in accordance with the teachings of the invention;

FIG. 5 is an elevational view of sedimentation rack means utilized in the practice of the invention;

FIG. 6 is a front elevational view with parts in cross section of magnetic separator and sample transfer apparatus constructed and operative in accordance with the teachings of the invention;

FIG. 7 is a rear elevational view of the magnetic separator portion of the apparatus of FIG. 6;

FIG. 8 is a top plan view of the magnetic separator of FIG. 7; and

FIG. 9 is an elevational view of the sample transfer tube.

DETAILED DESCRIPTION OF THE INVENTION Whole human blood comprises a mixture of red blood cells or erythrocytes, white blood cells or leukocytes, platelets, and the blood plasma or serum, respectively. The said white blood cells in turn comprise a cellular mixture of, for example, approximately 63% to 72% of neutrophils, approximately 2% to of eosinophils, approximately 0% to 1% of basophils, approximately 4% to 8% of monocytes, and approximately 20% to 30% of lymphocytes, it being understood that these figures are generally applicable to the majority of healthy adults.

It is the overall function of the new and improved method and apparatus of this invention to effect the separation of said lymphocytes from samples of whole human blood with very high combined degrees of purity, yield and viability. Purity may be understood to be equal to the number of lymphocytes present in the process output as divided by the total number of white blood cells or leukocytes present therein; while yield may be understood to be equal to the number of lymphocytes per cubic millimeter of output as divided by the number of lymphocytes per cubic millimeter of the original whole blood sample. Viability may be understood to be equal to the number of living lymphocytes in the output as divided by the total number of lymphocytes in said output.

In accordance with the teachings of this invention, the lymphocyte separation is effected for each whole blood sample by the tagging of the phagocytic leukocytes, or neutrophils, monocytes, eosinophils and basophils, respectively, of a whole blood sample with sensitized mag netic particles to the substantial exclusion of the lymphocytes, the settling and removal of the major portion of the erythrocytes, and the magnetic separation of the thusly tagged phagocytic leukocytes, along with the majority of the platelets which will cling thereto, from the whole blood sample leaving substantially all of the lymphocytes with the residual erythrocytes and blood plasma for subsequent lymphocyte extraction therefrom by simple hemolysis and washing or the like.

In accordance with the teachings of this invention, the whole blood sample is taken from the patient by the direct hypodermic needle withdrawal thereof into a Vacutainer tube, as manufactured and marketed by Becton, Dickinson & Company of Rutherford, N.J., in which is present a predetermined measured quantity of a suitable physiological stabilizer and anti-coagulant in the nature of the disodium or tripotassium salts Off the chelating agent EDTA, to result in the substantially immediate formation of what maybe termed EDTA blood to physiologically stabilize the white blood cells or leukocytes of the whole blood sample and prevent clotting of the red blood cells or erythrocytes to very significant advantage. More specifically, it may be understood that the leukoadhesive, phagocytic and clumping functions of the phagocytic leukocytes which provide for the tagging thereof by the sensitized magnetic particles, require the presence of free positive ions in the nature of calcium and magnesium ions for the occurrence thereof. The immediate formation as described of the EDTA blood results in the binding of these free positive ions by the EDTA with the result that said ions are no longer available in free form to thus substantially inhibit said leukoadhesive, phagocytic and clumping functions at this point in time. This is to say that, in the absence of the addition of the chelating agent in the nature of EDTA to the whole blood sample as described, or the alternative costly and inconvenient refrigeration of the whole blood sample to a low temperature between the time the same is withdrawn from the patient and the time the lymphocites are to be separated therefrom, the leukoadhesive, phagocytic and clumping functions of the phagocytic leukocytes will occur unchecked with resultant whole blood cell destruction and attendant significant decrease in cell viability. This will in turn result in major contamination of the lymphocytes by the dead cells which will then function to substantially inhibit the subsequent leukoadhesion and phagocytosis as must, of necessity, occur to effect lymphocyte separation in accordance with the teachings of the invention with high degrees of purity, yield and viability. Alternatively, it may be understood that said physiological stabilizer and anti-coagulant may, [for example, take the form of the sodium salts of CHEL DPTA and CHEL DM acid.

The separation of the lymphocites from the EDTA blood is initiated by the mixture thereof with a separating agent containing the following constituents to form a whole blood sample-separating agent mixture in the manner described in detail hereinbelow:

(a) Magnetic particles such as carbonyl ion particles, ferrite particles or magnetite particles in the size range of one to four microns which function, following the sensitizing thereof as described in detail hereinbelow, to effect the tagging of the phagocytic leukocytes and enable the subsequent magnetic sweeping thereof from the erythrocytes, blood plasma and lymphocytes with minimum entrapment of the latter.

(b) Magnesium and calcium free ions in the form of dissolved salts of calcium chloride and magnesium chloride which function to restore the ionic content of the EDTA blood to its original value by the replacement of the positive free ions bound as described hereinaboveby EDTA to thereby provide an optimum ionic concentration for leukoadhesion and phagocytosis.

(c) A minimum amount of a suitable anti-coagulant in the nature of heparin to insure the prevention of blood sample clotting during the time required for the lymphocyte separation process.

(d) A sensitizing agent comprising positively charged molecules of, for example, a basic poly amino acid or polypeptide in the nature of poly L lysine, polybrene, poly D lysine, poly DL lysine, or polyarginine or the like to enhance leukoadhesion and phagocytosis by sensitizing or increasing the positive surface charge on said magnetic particles through adsorption thereto.

(e) A dextrose solution to provide energy for the phagocytosis process.

(f) An isotonic solution in the nature of Hanks BSS of the same osmatic pressure as blood plasma to provide a physiological solution medium for the separation process.

(g) A red cell sedimenting agent comprising a high molecular weight settling agent in the nature, for example, of Dextran, Ficoll, PHA or the like in combination with, for example, a small amount of the monosodium or disodium salts of EDTA to promote erythrocyte sedimentation by causing the same to aggregate and thus fall more readily to the bottom of the mixture stream.

The separation of the lymphocytes from the whole blood sample in accordance with the teachings of this invention is then effected by the incubation of the resultant whole blood sample-separating agent mixture to promote phagocytic leukocyte tagging by the sensitized magnetic particles, to the substantial exclusion of the lymphocytes, through leukoadhesion, phagocytosis and clumping, respectively, the aggregation, settling and re moval of the major portion of the erythrocytes, and the subsequent magnetic sweeping of the thusly tagged phagocytic leukocytes, and the majority of the platelets from the whole blood sample.

The exact manners in which leukoadhesion, phagocytosis and leukocyte clumping take place are shown and described in detail in the copending application of myself and others entitled New And Improved Method And Apparatus for Lymphocyte Separation From Blood Ser. No. 79,913 filed Oct. 12, 1970, and assigned to the as signee hereof.

Referring now to FIG. 1, a sterile, disposable syringe is indicated at 10 and comprises a calibrated, transparent body part 12, a plunger 14 and a Luer-Lok tip 16. The syringe '10 preferably takes the form of the Plastipak as manufactured and marketed by Becton, Dickinson and Company of Rutherford, N].

A separating agent container is indicated at 18 in FIG. 2 and comprises a readily puncturable top 20. A supply of the separating agent discussed hereinabove is contained in container 18 as indicated at 22. A Vacutainer tube is indicated at 24 in FIG. 3 and a Whole human blood sample which has been fixed as described hereinabove is contained therein as indicated at 26, it being understood that said blood sample was withdrawn directly from the donor into said Vacutainer in conventional manner. The Vacutainer tube 24 is sealed by a readily puncturable top 27.

In use, the Luer-Lok tip cap is removed from syringe and a sterile hypodermic needle attached to the latter in conventional manner. This needle is then forced through the puncturable top of the separating agent container 18 into the separating agent 22 whereupon a measured amount of the latter is withdrawn into said syringe 10 by manipulation of the plunger 14 in conventional manner. Following this the syringe needle is inserted into the fixed whole blood sample 26 in the Vacutainer 24 and substantially all of the former is then drawn into the syringe 10 to mix therein with the separating agent. Preferably, a measured quantity of ambient air is then drawn into the syringe 10 to insure proper blood sample-separating agent mixture. Following this, the syringe needle is preferably re-capped to prevent leakage and insure continued sterility.

All of the above having been accomplished, the new loaded syringe is operatively positioned in incubatorrotary mixer means as indicated generally at 28 in FIG. 4 and which comprise a support frame 30 from which is supported a driven shaft 32. The shaft 32 is drivingly rotated in the clockwise direction as indicated by any convenient source of power in the nature of non-illustrated electric motor means which are also supported from the support frame 30. A generally triangular shaped support arm 34 is fixedly secured to shaft 32 so as to be rotatable therewith. The support arm 34 comprises syringe mounting means which may take the form of a conventional clamp as indicated at 36 and which are operable to secure the syringe 10 thereto as shown to thus operatively position the latter in the incubator-rotary mixer means 28. The support arm 34 is preferably designed so that the same will stop on the position depicted in FIG. 4 with the syringe 10 substantially vertical when driven rotation of shaft 32 is terminated.

A housing 38 surrounds support frame 30, and ambient air heating and supply means are schematically indicated at 48 and operatively connected as shown with said housing to provide a supply of air at substantially 37 C. thereto for blood sample separating agent mixture incubation within said housing.

Alternatively, the incubator means may be constructed separately and the rotary mixer simply placed therein in conventional manner.

With the loaded syringe 10 operatively positioned as described in the incubator-rotary mixer means 28 and air heating and supply means 40 operating to maintain the temperature within housing 38 at substantially 37 C., it may be understood that driven rotation of shaft 32 at, for example, approximately 5 rpm. for, for example, approximately 30 minutes, will be effective to insure excellent blood sample-separating agent mixture and incubation with attendant tagging of the phagocytic blood sample leukocytes by leukoadhesion, phagocytosis and clumping as described hereinabove.

Following this, the syringe 10 is preferably removed from the incubator-rotary mixer means 28 and placed as shown in the sedimentation rack 42 of FIG. 5 and allowed to stand for, for example, approximately 30 minutes to provide for erythrocyte sedimentation to the lower or plunger end of the syringe 10. Alternatively, the syringe 10 may, of course, simply be left in situ in the incubator rotary mixer means 28 for erythrocyte sedimentation. Incubation is, in any event, continued during sedimentation which, for use of the rack 42, would involve the place ment thereof in suitable, non-illustrated incubator means.

At the completion of mixture sedimentation, the syringe 10 is operatively positioned in magnetic separator and mixture transfer means as indicated generally at 44 in FIGS. 6, 7 and 8. Said magnetic separator and mixture transfer means com-prise a support frame 46 having clamping means 48'and guides 50 for operative positioning of the syringe 10 thereon as shown.

A syringe plunger drive shaft is indicated at 52 and extends outwardly as shown from an elongated slot 54 which is formed in the support frame 46. The drive shaft 52 abuts the bottom of the syringe plunger 14 and is drivable to reciprocate as indicated in the slot 54 under the control of non-illustrated electric drive motor means.

Magnetic separator means are indicated generally at 56 in FIGS. 6, 7 and 8 and comprise a generally U shaped, high field density magnet 58 which may, for example, take the form of an Alnico magnet, and which is supported as shown from the rear side of the support frame 46 by

non-magnetic supports

60 and 62. Soft steel pole pieces are indicated at 64 and 66 and are secured as shown by any suitable manner to the respective extremities of the magnet 58. A soft steel core piece is indicated at 68 and is supported as shown (FIG. 7) from the

respective pole pieces

64 and 66 by a support assembly 70 of any suitably non-magnetic material. An aluminum guide piece is indicated at 72 and is affixed as shown in any convenient manner to the core piece 68.

The respective inner edges of the

pole pieces

64 and 66 are cut-out in generally arcuate manner as best seen in FIG. 8 to remain substantially equally spaced from the periphery of the generally circular core piece 68. In addition, the respective upper edges of said pole and core pieces, and the lower edge of the generally circular guide piece 72 are cut back in generally arcuate manner as best seen in FIG. 7 to provide a generally circular mounting groove which extends substantially around said guide and core pieces.

A blood sample-separating agent mixture transfer tube is indicated at 74 in FIG. 9 and is preferably constituted by an appropriate length of sterile PVC tubing which is sealed as shown at 76 and 78 adjacent its respective extremities to provide a sterile, enclosed transfer volume 80. The transfer tube 74 is operatively positioned in the magnetic separator and mixture transfer means by placing the sealed tube extremity 78 in the notch provided therefor by tube support fixture 82 (FIG. 6), passing the tube through tube guide notch 84 in support frame 46, to the rear of the latter, passing the tube in under-over manner around the core piece 68 in the groove provided therefor as described by said core piece, the

pole pieces

64 and 66 and the guide piece 72 as best seen in FIG. 8, returning the tube to the front side of the support frame 46 through tube guide notch 86, passing the tube underneath guide post 88, and fastening the sealed tube extremity 76 in the notch provided therefor in tube support fixture 92, all as best seen in FIGS. 6, 7 and 8.

A receptor tube assembly is indicated generally at in FIG. 6 and comprises a receptor tube 92 which fits tightly as shown into an adapter 94. The receptor tube is preferably constituted by a stoppered evacuated Vacutainer tube. A hypodermic needle 96 extends as shown through the top 98 of the adapter 94 and, upon insertion of the receptor tube 92 thereinto, functions to puncture the readily punctuable cap 100 of the latter. A bent hypodermic needle or similar small conduit 102 extends as shown through the receptor tube adapter top 98 through the receptor tube cap 160, and extends from the former to atmosphere. A micro filter is schematically indicated at 104 and is disposed as shown in codnuit 102 to thoroughly filter any air which may fiow therethrough into receptor tube 92 upon the initial puncturing of the cap of the latter.

With the assembled receptor tube 92 and adapter 94 positioned in

guides

106, 108 and 110 on the support frame 46, it may be understood that upward movement thereof will effect the puncturing of the transfer tube 74 by hypodermic needle 96 adjacent tube seal 76 whereupon the receptor tube assembly may be locked in the depicted position thereof by actuation of the clamping means 112.

With the transfer tube 74 and the receptor tube assembly 90 operatively positioned as described in the magnetic separator and mixture transfer means 44, it may be understood that the loaded syringe is operatively positioned in the former as described to effect the puncture of said transfer tube adjacent the sealed tube extremity 74. This having been done, and with plunger drive shaft 52 positioned in slot 54 at a location commersurate with the bottom of the still withdrawn syringe plunger 14, it may be understood that operation of the magnetic separator and mixture transfer means 44 is started by commencing the upward movement of said drive shaft at constant rate which, for use as described in the separation of the lymphocites from whole blood samples, will be that rate sufiicient to provide for a blood sample-separating agent mixture pumping or flow rate of approximately 3.5 ml./min. into said transfer tube.

As the mixture of interest flows into and through the transfer tube 74 and around the core piece 68 it may be understood that the magnetic particles, along with the leukocytes which have been tagged thereby by leukoadhesion, phagocytosis and clumping as described in detail hereinabove, will be subjected to the high intensity, nonuniform magnetic field set up in the magnetic separator 56 as illustrated by the lines of force drawn in FIG. 7 to strongly pull said magnetic particles downwardly as shown to the bottom of the flowing stream and retain the same within that portion of the transfer tube which winds through said magnetic separator. Calculations have established that with a magnetic field of sufiicient strength to create a downward force of approximately 1000 Gs on said magnetic particles, and with a transfer tube passage length of approximately 3.5 inches through the magnetic separator 56 and a mixture flow rate of approximately 3.5 ml./min., very close to 100% retention of the magnetic particles, and .thus effective removal thereof along with the leukocytes tagged thereby from the flowing stream may be achieved.

The pumping as described of the blood sample-separating agent mixture from the syringe 10 into the transfer tube 74 will, of course, result in an increase in pressure in the latter whereby that portion of the flowing stream which exits from the magnetic separator 56 to the extremi ty of said transfer tube at seal 76 will be pressure pumped from said extremity into and through hypodermic needle 96 into the receptor tube 92. This portion of the flowing stream which is pumped as described into the receptor tube 92 will contain, in suspension in the blood plasma, a very high percentage of the live lymphocytes from the whole blood sample of interest, plus a small residual amount of the erythrocytes and a very small measure of contaminating cells in the nature of platelets, eosinophils and/ or basophils.

Operation continues as described until such time as the erythrocytes or red cells which had been substantially settled by sedimentation to the lower portion of the syringe body 12 are observed to commence arrival just beyond that portion of the generally transparent transfer tube 74 which underlies the guide post 88 as best seen in FIG. 6, whereupon pumping is discontinued by termination of the upward movement of plunger drive shaft 52. Thus is insured that no part of the major portion of the erythrocytes which had been settled to the lower portion of the syringe 10 will be pumped into the receptor tube 92.

At the completion of the pumping operation, the rec pt r t be 92 i removed f om he pport frame. 46

and receptor tube adapter 94 in obvious manner whereupon separation of the live lymphocytes from the thusly collected lymphocyte-erythrocyte contaminating cellsplasma suspension may be readily and efficiently effected by simple hemolysis and subsequent lymphocyte wash and resuspension in manner well known to those skilled in this art. Operation for a representative whole blood sample may be understood to take approximately 10 minutes.

Utilization of the method and apparatus for succeeding whole blood samples is effected in the manner. described, it being understood that a new, and accordingly completely sterile, syringe, transfer tube, and receptor tube including a new hypodermic needle 96, is utilized for each sample. As a result, absolutely sterile conditions are provided for each blood sample whereupon inter-sample contamination of the separated lymphocytes of interest is absolutely prevented to very significant advantage as should be obvious.

Actual utilization of the method on apparatus of the invention for the separation as described of the lymphocytes from whole blood samples has proven effective to provide for such separation with a combination of, on the average, approximately 97% purity, approximately yield, and approximately viability.

Although disclosed hereinabove as applied to the separation of lymphocytes from blood samples it may be understood that the method and apparatus of the invention are also applicable to the separation of other and different constituents from said blood samples providing, of course, that said constituents are separable therefrom attendant the operation of magnetic separator means. Also, and subject of course to the same qualifications, it is believed clear that the method and apparatus of the invention are applicable to the separation of constituents from samples other and different than blood samples.

While there has been shown and described the preferred embodiment of the invention, it will be understood that the invention may be embodied otherwise than as herein specifically illustrated or described, and that certain changes in the form and arrangement of parts and in the specific manner of practicing the invention may be made without departing from the underlying idea or principles of this invention within the scope of the appended claims.

What is claimed is:

1. In apparatus for the separation of the lymphocytes from a blood sample, the improvements comprising, means to collect and mix said blood sample and a separating agent which includes sensitized magnetic particles, means to incubate the resultant separating agent-blood sample mixture to effect the tagging of the blood sample leukocytes by said sensitized magnetic particles to the substantial exclusion of the lymphocytes through leukoadhesion, phagocytosis and clumping, respectively, transfer means, magnetic separating means operatively associated with said transfer means, lymphocyte collecting means, means to operatively connect said blood sample and separating agent collecting means, said transfer means and said lymphocyte collecting means for flow therebetween, and means to flow said blood sample-separating agent mixture from said blood sample and separating agent collecting means through said transfer and operatively associated magnetic separating means for substantial retention of said tagged leukocytes in the latter and continued flow of said lymphocytes through said transfer means into said lymphocyte collection means.

2. In apparatus as in claim 1 wherein, said blood sample and separating agent collecting means comprise a syringe.

3. In apparatus as in claim 1 wherein, said transfer means comprise tubing.

4. In apparatus as in claim 1 wherein, said transfer means comprise a length of tubing which is sealed adjacent each extremity thereof, said blood sample and separating agent CQlleQting means comprise a syringe having a hypodermic needle extending therefrom, said lymphocyte collecting means comprise a container having a hypodermic needle extending therefrom, and said syringe and container are operatively connected to said tubing by extension of the respective hypodermic needles thereof into the sealed interior of said tubing at spaced locations on the latter.

5. In apparatus as in claim 1 wherein, said separating agent further includes an erythrocyte sedimenting agent, and said apparatus further includes sedimentation means for sedimentation of said blood sample-separating agent mixture for settling of the erythrocytes.

6. In apparatus as in claim 3 wherein, said magnetic separating means comprise means defining a generally arcute passage through which is passed a high density,

UNITED STATES PATENTS 3,650,698 3/1972 Adler 23-230 B X LIONEL M. SHAPIRO, Primary Examiner R. J. WARDEN, Assistant Examiner US. Cl. X.R.

l95l.8; 23230 B