Concentrating White Blood Cells for DNA Extraction from a Leukodepleted Blood Sample
Reagents and a method for the pre-concentration of WBCs from leukodepleted blood segment samples are described. The reagent comprises: a lytic reagent, for example, saponin in phosphate buffered saline(PBS), wherein the amount of saponin is in the range of from about 1% to about 10% percent (w/w). The method comprises: contacting a specific volume the leukoreduced blood segment sample with a specific volume of the lytic regent; mixing the two so as to selectively lyse RBCs in the leukoreduced whole blood segment sample; subjecting the mixture to centrifugation, so as to separate the mixture into a WBC rich phase and a lysed-RBC phase; discarding the supernatant containing the lysed RBC phase; and resuspending the WBC rich phase in a specific volume of PBS.
1 . A method for extracting genomic DNA from leukoreduced blood samples, the method comprising:
contacting a specific volume the leukoreduced blood sample with a specific volume of a lytic reagent capable of lysing red blood cells;
mixing the two so as to selectively lyse red blood cells in the leukoreduced blood sample;
subjecting the lysed mixture to centrifugation, so as to separate the mixture into a white blood cell rich sediment and a supernatant rich in lysed-red blood cells;
discarding the supernatant;
resuspending the white blood cell rich phase in a specific volume of aqueous buffer; and
extracting DNA from the buffered white blood cell suspension.
2 . The method of claim 1 , wherein the lytic reagent comprises saponin dissolved in phosphate buffered saline.
3 . The method of claim 2 wherein the amount of saponin in the lytic reagent is from about 1% to about 10% percent (w/w).
4 . The method of claim 2 wherein the lytic reagent further includes alkali and the pH is near neutral.
5 . The method of claim 1 , wherein the volume of the leukoreduced blood sample is about 0.2 ml to about 2 ml.
6 . The method of claim 5 , wherein the volume of the lytic reagent is about 40 μl to about 80 μl.
7 . The method of claim 1 , wherein the buffer is selected from the group consisting of hydrogenated phosphates of sodium and potassium.
8 . The method of claim 1 , wherein the centrifugation is done at about 300×g to 2000×g and for about 2 to 6 minutes.
9 . The method of claim 1 wherein the centrifugation is at 2000×g for 3 minutes.
10 . The method of claim 1 , wherein the volume of buffer used for WBC resuspension is from about 50 μl to about 500 μl.
11 . The method of claim 1 wherein the resuspension of the white blood cell rich phase in phosphate buffered saline is by vortexing.
12 . The method of any of claims 1 to 3 further comprising the step of isolating genomic DNA from the white blood cell rich phase.