Methods for isolating target cells from blood

Abstract

Disclosed herein are methods for isolating target cells from blood, involving mixing in an open container an undiluted blood sample having a volume of 10 ml or less, and binding agents, wherein each binding agent comprises (A) a primary binding agent comprising an agent capable of binding to at least one cellular epitope on target cells in the undiluted blood sample, (B) a first linker bound to the primary binding agent, to generate binding agent-attached target cells in the undiluted blood sample; contacting the binding agent-attached target cells in the undiluted blood sample with a plurality of buoyant reagents that include a second linker capable of binding to the first linker to generate an undiluted buoyant reagent-attached target cell mixture; diluting the undiluted buoyant reagent-attached target cell mixture by at least 20% to produce a diluted buoyant reagent-attached target cell mixture; applying a vectorial force, such as centrifugal force, to the diluted buoyant reagent-attached target cell mixture to generate a stratified diluted buoyant reagent-attached target cell mixture; removing the buoyant reagent-attached target cells from the stratified diluted buoyant reagent-attached target cell mixture; and isolating the target cells from the buoyant reagent-attached target cells.

We claim: 
     
         1 . A method for isolating target cells from blood, comprising:
 (a) mixing in an open container
 (i) an undiluted blood sample having a volume of 10 ml or less, and 
 (ii) binding agents, wherein each binding agent comprises (A) a primary binding agent comprising an agent capable of binding to at least one cellular epitope on target cells in the undiluted blood sample, (B) a first linker bound to the primary binding agent, 
   wherein the mixing occurs for a time and under conditions suitable to promote binding of the primary binding agents to the target cells to generate binding agent-attached target cells in the undiluted blood sample;   (b) contacting the binding agent-attached target cells in the undiluted blood sample with a plurality of buoyant reagents, wherein each buoyant reagent comprises a second linker bound to the buoyant reagent, wherein the second linker is capable of binding to the first linker, wherein the contacting occurs for a time and under conditions suitable to promote binding of the second linker to the first linker to generate an undiluted buoyant reagent-attached target cell mixture;   (c) diluting the undiluted buoyant reagent-attached target cell mixture by at least 20% to produce a diluted buoyant reagent-attached target cell mixture;   (d) applying a vectorial force, such as centrifugal force, to the diluted buoyant reagent-attached target cell mixture to generate a stratified diluted buoyant reagent-attached target cell mixture;   (e) removing the buoyant reagent-attached target cells from the stratified diluted buoyant reagent-attached target cell mixture; and   (f) isolating the target cells from the buoyant reagent-attached target cells.   
     
     
         2 . The method of  claim 1 , wherein the buoyant reagents comprise gas-filled bubbles. 
     
     
         3 . The method of  claim 2 , wherein the gas-filled bubbles comprise perfluorocarbon gas cores encompassed by lipid or phospholipid shells. 
     
     
         4 . The method of  claim 2 , wherein the gas-filled bubbles have a mean size volume of greater than 6 μm 3  and less than 10 μm 3 . 
     
     
         5 . The method of  claim 2 , wherein the gas-filled bubbles have a mean size diameter of between 1.5 μm and about 3 μm. 
     
     
         6 . The method of  claim 2 , wherein the gas-filled bubbles are present in the contacting step at a concentration of at least 4×10 8  per ml. 
     
     
         7 . The method of  claim 1 , wherein the second linker comprises streptavidin (SA), and the first linker comprises biotin. 
     
     
         8 . The method of  claim 7 , wherein the streptavidin is present on the buoyant reagent at a density of greater than 20,000 molecules per um 2 . 
     
     
         9 . The method of  claim 7 , wherein the streptavidin is present on the buoyant reagent at a density of greater than 25,000 molecules per um 2 . 
     
     
         10 . The method of  claim 7 , wherein the streptavidin is present on the buoyant reagent at a density of greater than 26,000 molecules per um 2 . 
     
     
         11 . The method of  claim 1 , wherein the binding agents comprise antibodies attached to the first linker. 
     
     
         12 . The method of  claim 1 , wherein the undiluted blood sample has a volume of 5 ml or less. 
     
     
         13 . The method of  claim 1 , wherein the undiluted blood sample has a volume of 3 ml or less. 
     
     
         14 . The method of  claim 1 , wherein the undiluted blood sample has a volume of 1 ml to 3 ml. 
     
     
         15 . The method of  claim 1 , wherein the diluting comprises diluting the undiluted buoyant reagent-attached target cell mixture by between 20% and 500% to produce the diluted buoyant reagent-attached target cell mixture. 
     
     
         16 . The method of  claim 1 , wherein the removing step comprises removing the buoyant reagent-attached target cells from the stratified diluted buoyant reagent-attached target cell mixture using a pipette or syringe. 
     
     
         17 . The method of  claim 1 , wherein the isolating step comprises isolating the target cells from the buoyant reagent-attached target cells by sonicating or applying positive pressure to degas the microbubbles. 
     
     
         18 . The method of  claim 1 , wherein the target cells comprise cells of surface immunophenotypes comprising one or more of CD45+, CD3+, CD4+, CD8+, CD25+, CD14+, CD16+, CD19+, CD56+, CD34+, CD117+, CD235a, CD349 − , T cell receptor (TCR) alpha, gamma, beta & delta. 
     
     
         19 . The method of  claim 1 , wherein the target cells comprise CD3 +  cells. 
     
     
         20 . The method of  claim 1 , wherein the target cells are isolated with a purity of at least 85%. 
     
     
         21 . The method of  claim 1 , wherein the target cells are isolated with a viability of at least 80%. 
     
     
         22 . The method of  claim 1 , wherein at least 50% of the target cells in the blood sample are isolated. 
     
     
         23 . The method of  claim 1 , wherein the target cells are not platelets or red blood cells.