Stabilization of whole blood at room temperature

Abstract

The present disclosure is directed to the stabilization of nucleated cells in a whole blood sample ex-vivo, effected by an additive being a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant, preferably a mitochondria-targeted antioxidant selected from the group consisting of SkQ1, MitoQ, SS-31, and a mixture thereof. In a specific embodiment, the liquid composition further comprises a protease inhibitor. Further provided is advantageous use of the composition for stabilizing an analyte selected from DNA, RNA and protein in intact nucleated cells of the whole-blood sample ex-vivo, as well as kits including the composition for practicing said use. Additionally, specific methods are provided for stabilizing intact nucleated cells of a whole-blood sample ex-vivo.

1 . A liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant. 
     
     
         2 . The composition of  claim 1  wherein the mitochondria-targeted antioxidant is selected from the group consisting of SkQ1, MitoQ, SS-31, or a mixture thereof. 
     
     
         3 . The composition according to  claim 1 , wherein the composition further comprises a protease inhibitor. 
     
     
         4 . The composition of  claim 1 , wherein the antioxidant is a mixture of a first and a second antioxidant, wherein the first antioxidant is a mitochondria-targeted antioxidant, and the second antioxidant is an antioxidant other than a mitochondria-targeted antioxidant. 
     
     
         5 . The composition of  claim 4 , wherein the second antioxidant is selected from the group consisting of glutathione, acetylsalicylic acid, N-acetyl-5-aminosalicylic acid, N-acetylcysteine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and a mixture thereof. 
     
     
         6 . The composition according to  claim 4 , wherein the first mitochondria-targeted antioxidant is selected from the group consisting of SkQ1, MitoQ, SS-31, and a mixture thereof. 
     
     
         7 . The composition according to  claim 1 , wherein the cell-metabolizable sugar is selected from the group consisting of glucose and fructose. 
     
     
         8 . The composition according to  claim 1 , the composition being enclosed in a blood drawing container. 
     
     
         9 . The composition according to  claim 1 , further comprising an amount of whole blood. 
     
     
         10 . The composition according to  claim 9 , wherein the composition comprises one or more mitochondria-targeted antioxidant(s) with an aggregate concentration of 10 nM to 200 μM. 
     
     
         11 . The composition according to  claim 10 , wherein the composition comprises a concentration of a mitochondria-targeted antioxidant selected from the group consisting of 50 nM-250 nM. 
     
     
         12 . The composition according to  claim 11 , wherein the composition comprises 11.1 mM-12.3 mM trisodium citrate, 2 mM-2.2 mM citric acid, 2 mM-2.2 mM NaH 2 PO 4 , 19.1 mM-21.1 mM glucose, 0.23 mM-0.25 mM adenine, 0.24 mM-0.26 mM AEBSF, 990 nM-1.1 μM SS-31 and acetylsalicylic acid 990 μM-1.1 mM. 
     
     
         13 . A kit of parts comprising a composition according to  claim 8 , the kit further comprising packaging material, a label, and a user instruction sheet. 
     
     
         14 . A method for stabilizing intact nucleated cells of a whole-blood sample ex-vivo, the method comprising the steps of
 (a) providing the whole blood sample ex-vivo;   (b) contacting and mixing the sample of step (a) with a composition according to  claim 1 ;   (c) incubating the mixture obtained in step (b),   thereby stabilizing intact nucleated cells of the ex-vivo whole-blood sample.   
     
     
         15 . The method according to  claim 14 , wherein the mixture obtained in step (b) is a composition according to  claim 10 . 
     
     
         16 . The method according to  claim 14 , wherein step (c) is performed at room temperature for a time interval selected from the group consisting of 0 h-12 h, 0 h-24 h, 0 h-36 h, 0 h-48 h, 0 h-60 h, and 0 h-72 h. 
     
     
         17 . The method of  claim 14 , the method further comprising the steps of
 (d) separating intact nucleated cells from the incubated mixture obtained in step (c); and   (e) lysing the separated nucleated cells and detecting an analyte of nucleated cells in the lysate, wherein the analyte is selected from the group consisting of DNA, RNA, and protein.   
     
     
         18 . The method according to  claim 17 , wherein in the mixture of step (b) the mitochondria-targeted antioxidant is SS-31 at a concentration of 500 nM-10 μM. 
     
     
         19 . The method according to  claim 18 , wherein the mixture of step (b) further comprises acetylsalicylic acid at a concentration of 200 nM-10 mM, and AEBSF at a concentration of 50 nM-1 mM. 
     
     
         20 . The method according to  claim 14 , the method further comprising the steps of
 (d) separating intact nucleated cells from the incubated mixture obtained in step (c);   (e) contacting the separated nucleated cells of step (d) with a culture medium, and culturing viable cells.