Reagent composition for the analysis of residual white blood cells in leuko-reduced blood banking products
View Patent ↗The enumeration and analysis of residual white blood cells in a sample of leukocyte-reduced blood products is conducted by forming a suspension of the leukocyte-reduced blood products with a sufficient amount of a lysing reagent. The lysing reagent comprises a buffer with a low molar concentration, and a non-ionic surfactant. The suspension of leukocyte-reduced blood products and the lysing reagent is incubated for a sufficient time at a temperature sufficient to selectively lyse the platelets and red blood cells without damaging the white blood cells. The white blood cells of the lysed blood products are then contacted with a suitable dye to stain the white blood cells and the number of stained white blood cells is measured. The lysing reagent is free of harsh organic solvents which can damage the plastic components of automated clinical analyzers.
1. An automated clinical analysis method for the enumeration and analysis of residual white blood cells in a sample of leukocyte-reduced blood products selected from the group consisting of plasma, red blood cells, and platelets, consisting essentially of:
a) forming a suspension of said sample of leukocyte-reduced blood products by contacting said sample with a sufficient amount of a lysing reagent to selectively destroy the platelets and red blood cells in the sample, wherein said lysing reagent consists essentially of:
i) about 2mM to about 25mM of a buffer selected from the group consisting of succinic acid, citric acid, N-2-acetamidoiminodiacetic acid, 2-(N-morpholino)ethanesulfonic acid, and piperazine-N, N′-bis-(2-ethanesulfonic acid), and
ii) about 0.01 weight percent to about 1.0 weight percent of at least one non-ionic surfactant selected from the group consisting of octylphenol polyethylene glycol ether, polyethylene sorbitan monolaureate, polyethylene sorbitan monooleate, and polyoxyethylene lauryl ether, wherein the volumetric ratio of lysing reagent to sample of leukocyte-reduced blood products varies from about 2:1 to about 3:1 respectively;
b) incubating said suspension of leukocyte-reduced blood products and the lysing reagent for a sufficient time at a temperature sufficient to selectively lyse the platelets and red blood cells without damaging the white blood cells;
c) contacting the white blood cells of the lysed blood products with about 25 mg/L to about 200 mg/L of a nucleic acid specific dye to stain the DNA or nucleic acids within the nucleus of the white blood cells; and
d) aspirating the stained white blood cells to the direct cytometry feature of an automated clinical analyzer and measuring the number of stained white blood cells, based on their light absorption or light scattering characteristics; wherein said method is conducted at a pH of about 3.5 to about 5.5.
2. The method of claim 1 , wherein the non-ionic surfactant is octylphenol ethoxylate.
3. The method of claim 1 , wherein the buffer is 2-(N-morpholino) ethanesulfonic acid.
4. The method of claim 1 , wherein the nucleic acid specific dye is selected from the group consisting of methyl green, methylene blue, pyronin Y, and propidium iodide.
5. The method of claim 4 , wherein the nucleic acid specific dye is selected from the group consisting of methyl green and methylene blue.
6. The method of claim 1 , wherein the amount of nucleic acid specific dye varies from about 75 mg/L to about 125 mg/L of lysing reagent.
7. The method of claim 1 , wherein the concentration of white blood cells is measured in an automated hematology analyzer operating as a flow cytometer.
8. The method of claim 7 , wherein the incubated sample of leukocyte-reduced blood products is aspirated through a direct cytometry hydraulic pathway of an automated hematology analyzer.
9. The method of claim 1 , wherein the white blood cells are measured per unit volume of blood in the sample.