EP0190488A1
Expired / Lapsed
EP
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Separation of lymphocytes and monocytes from blood samples
Assignee: Becton Dickinson and Co
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Filing: 1985-11-05
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Granted: —
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Expires: 2005-11-05
Abstract
Abstract This invention is directed to means for inhibiting the apparent shift in the buoyant density of and/or to restore any loss in the buoyant density of the granulocytic white blood cells in a sample of blood, thereby insuring the quality of the separation of lymphocytes and monocytes from granulocytes in a blood sample. The invention comprises contacting a blood sample with a hypertonic fluid containing a low molecular weight organic or inorganic ionic substance and/or with an isotonic or hypertonic fluid containing a high molecular weight organic substance which may contain organic molecules having a lipophilic substituent in their structures and/or with a culture medium for blood cells. An integral part of the invention is an improved blood separation tube utilizing a gel-like substance having a specific gravity between 1.060-1.065 g/cm 3 to significantly enhance the purity of cell separation, while providing acceptable cell yields.
Inventors
- 👤 Richard John Carroll
- 👤 Ward Curtis Smith
- 👤 Albert August Luderer
- 👤 Anthony Ralph Zine
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-08 02:01
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/483; G01N33/487; G01N33/49; G01N33/491; Y; Y10; Y10T; Y10T436/00; Y10T436/25; Y10T436/25375
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
188 words
A method for separating lymphocytes and monocytes from granulocytes in a sample of anticoagulated blood wherein an apparent shift in the buoyant density of the granulocytes is inhibited and/or any loss in buoyant density of the granulocytes is restored which comprises the steps of: (a) mixing said blood sample with a hypertonic fluid containing a low molecular weight organic or inorganic ionic substance which is essentially chemically compatible with the blood cells and/or with an isotonic or hypertonic fluid containing a high molecular weight organic substance which is essentially chemically compatible with the blood cells and/or with a culture medium for blood cells, contact between said blood sample and said fluid being maintained for at least about 10 minutes; (b) placing a water-insoluble, thixotropic gel-like substance which is chemically inert to blood constituents and said fluid into said anticoagulated blood-fluid mixture; (c) centrifuging said blood-fluid-gel mixture at a force and for a sufficient length of time to cause said gel-like substance to flow sufficiently to form a barrier between the lymphocytes and monocytes and the granulocytes; and then (d) removing the lymphocytes and monocytes from atop said barrier.
Claim 2
Independent Claim
188 words
A method for separating lymphocytes and monocytes from granulocytes in a sample of anticoagulated blood wherein an apparent shift in the buoyant density of the granulocytes is inhibited and/or any loss in buoyant density of the granulocytes is restored which comprises the steps of: (a) mixing said blood sample with a hypertonic fluid containing a low molecular weight organic or inorganic ionic substance which is essentially chemically compatible with the blood cells and/or with an isotonic or hypertonic fluid containing a high molecular weight organic substance which is essentially chemically compatible with the blood cells and/or with a culture medium for blood cells, contact between said blood sample and said fluid being maintained for at least about 10 minutes; (b) placing a water-insoluble, thixotropic gel-like substance which is chemically inert to blood constituents and said fluid into said anticoagulated blood-fluid mixture; (c) centrifuging said blood-fluid-gel mixture at a force and for a sufficient length of time to cause said gel-like substance to flow sufficiently to form a barrier between the lymphocytes and monocytes and the granulocytes; and then (d) removing the lymphocytes and monocytes from atop said barrier.
Claim 3
Independent Claim
108 words
A method for inhibiting the apparent shift in the buoyant density of and/or to restore loss in the buoyant density of the granulocytic white blood cells in a sample of blood, thereby insuring a high quality separation of lymphocytes and monocytes from granulocytes in a blood sample, which comprises contacting said sample of blood with a hypertonic fluid containing a low molecular weight organic or inorganic ionic substance which is essentially chemically compatible with the blood cells and/or with an isotonic or hypertonic fluid containing a high molecular weight organic substance which is essentially chemically compatible with the blood cells and/or with a culture medium for blood cells.
Claim 4
Independent Claim
108 words
A method for inhibiting the apparent shift in the buoyant density of and/or to restore loss in the buoyant density of the granulocytic white blood cells in a sample of blood, thereby insuring a high quality separation of lymphocytes and monocytes from granulocytes in a blood sample, which comprises contacting said sample of blood with a hypertonic fluid containing a low molecular weight organic or inorganic ionic substance which is essentially chemically compatible with the blood cells and/or with an isotonic or hypertonic fluid containing a high molecular weight organic substance which is essentially chemically compatible with the blood cells and/or with a culture medium for blood cells.
Claim 5
Dependent Claim
27 words
A method as claimed in claim 2 characterised in that contact between said sample of blood and said fluid is maintained for more than approximately 1 minute.
Claim 6
Independent Claim
29 words
A method as claimed in any of claims 1 to 3 characterised in that said hypertonic fluid containing a low molecular weight ionic substance is an aqueous saline solution.
Claim 7
Independent Claim
26 words
A method as claimed in any of claims 1 to 3 characterised in that said ionic substance has a molecular weight of less than about 1500.
Claim 8
Independent Claim
29 words
A method as claimed in any of claims 1 to 5 characterised in that said high molecular weight organic substance contains molecules having a lipophilic substituent in their structure.
Claim 9
Dependent Claim
23 words
A method as claimed in claim 6 characterised in that said high molecular weight organic substance is metrizoic acid and/or a derivative thereof.
Claim 10
Dependent Claim
22 words
A method as claimed in claim 7 characterised in that said derivative of metrizoic acid is selected from sodium metrizoate and metrizamide.
Claim 11
Independent Claim
22 words
A method according to any of claims 1 to 5 characterised in that said high molecular weight organic substance is N,N'-Bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl) acetamido]-2,4,6-triiodo-isophthalamide.
Claim 12
Independent Claim
34 words
A method as claimed in any of claims 1 to 9 characterised in that the culture medium for blood cells is selected from the group of Roswell Park Memorial Institute medium and McCoy's medium.
Claim 13
Independent Claim
26 words
A method as claimed in any of claims 1 to 10 characterised in that said gel-like substance has a specific gravity between 1.055-1.075 g/cm 3 .
Claim 14
Dependent Claim
23 words
A method as claimed in claim 11 characterised in that said gel-like substance has a specific gravity between 1.060-1.065 g /cm 3 .
Claim 15
Independent Claim
26 words
A method as claimed in any of claims 1 to 12 characterised in that said centrifuging is conducted at a force of at least 1200 G's.
References Cited (Backward Citations)
US4190535A
Cited
DE2917887A1
Cited
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