Methods for the isolation of nucleic acids and for quantitative DNA extraction and detection for leukocyte evaluation in blood products

A method for determining the number of cells of interest in a sample, wherein the cells of interest comprise white blood cells comprising DNA, the method comprising:

• (a) applying a sample comprising cellular material to a filter, wherein the cellular material comprises cells of interest;
• (b) lysing the cells of interest to form a cell lysate;
• (c) immobilizing the released DNA on or within the filter;
• (d) removing the remaining cell lysate from the filter and retaining the DNA;
• (e) quantifying the amount of the DNA retained on the filter;
• and
• (f) determining the number of cells of interest in the sample based on the determined quantity of the DNA retained.

A method for estimating the number of white blood cells in a blood sample from a source of interest, wherein the white blood cells comprise DNA, the method comprising:

• (a) isolating the DNA from the blood sample;
• (b) determining the amount of the DNA isolated from the blood sample;
• and
• (c) determining the number of white blood cells in the blood sample based on the determined amount of the isolated DNA.

A method for determining the amount of white blood cells in a blood sample, wherein the white blood cells comprise DNA, the method comprising:

• (a) applying a blood sample comprising cells to a filter in the absence of a chaotrope;
• (b) retaining the cells with the filter as a cellular retentate and removing contaminants;
• (c) lysing the cellular retentate from step (b) to form a cell lysate while retaining the cell lysate in the filter, the cell lysate retentate containing the DNA;
• (d) removing the remaining cell lysate from the filter and retaining the DNA;
• (e) eluting the DNA;
• (f) measuring the amount of the DNA eluted from the filter;
• and
• (g) determining the amount of the DNA measured to estimate the number of white blood cells in the sample based on an amount of approximately 7 pg of nucleic acid contained in each white blood cell.

A method for isolating DNA from a blood sample comprising:

• (a) applying a blood sample comprising cells to a filter in the absence of a chaotrope;
• (b) retaining the cells with the filter as a cellular retentate and removing contaminants;
• (c) lysing the cellular retentate from step (b) to form a cell lysate while retaining the cell lysate in the filter, the cell lysate retentate containing DNA;
• (d) removing the remaining cell lysate from the filter and retaining the DNA;
• (c) heating the DNA to an elevated temperature of 40° C. to 125° C. while retained by the filter;
• (f) eluting the DNA;
• (g) quantifying the amount of DNA eluted from the filter;
• and
• (h) determining the number of DNA-containing cells in the sample based on the determined quantity of the DNA eluted.

A method for estimating the amount of white blood cells in a leukoreduced blood sample, wherein the white blood cells comprise DNA, the method comprising:

• (a) compressing a filter to between 10% and 80% less than its original volume;
• (b) applying a leukoreduced blood sample comprising cells to the filter in the absence of a chaotrope;
• (c) retaining the cells with the filter as a cellular retentate comprising whole cells, condensed nuclear material, and cell debris;
• (d) removing non-cellular contaminants from the retentate;
• (e) lysing the cells in the cellular retentate from step (b) while the retentate is entrapped within the filter to form a cell lysate containing the DNA;
• (f) retaining the DNA while removing remaining cell lysate;
• (g) heating the DNA to an elevated temperature of 40° C. to 125° C. while retained by the filter,
• (h) eluting the DNA;
• (i) measuring the amount of genomic DNA eluted from the filter;
• (j) repeating steps (g) and (h);
• and
• (k) determining the amount of DNA measured to estimate the number of white blood cells in the sample based on an amount of approximately 7 pg of DNA contained in each white blood cell.

A kit for quantifying the amount of genomic DNA in a blood sample comprising cells containing DNA comprising:

• (a) a filter supported on a support, said filter comprising matrix means for physically retarding movement of DNA therethrough in the absence of a chaotrope;
• (b) one or more solutions selected from the group consisting of a red cell lysis solution, a solution for rupturing intact whole cells to leave condensed nuclear material, a lysis solution for lysing nuclear material and an elution solution;
• and
• (c) an indicator capable of providing quantitative measurement of double-stranded or genomic DNA in a concentration as low as 1000 ng/ml.

A method for estimating the amount of white blood cells in a blood sample, wherein the white blood cells comprise DNA, the method comprising:

• (a) applying a blood sample to a substrate consisting of a coating enabling cellular lysis and release of the DNA, wherein the coating comprises a surfactant or detergent;
• (b) immobilizing the released DNA to a matrix, the substrate physically capturing the DNA;
• (c) bonding the DNA to the substrate;
• (d) generating a signal when the DNA bonds to the substrate;
• (e) analyzing the amount of DNA captured by quantifying the generated signal;
• (f) estimating by calculation the number of white blood cells in the blood sample based on the amount of DNA isolated.