Enhancing flow cytometry discrimination with geometric transformation
In flow cytometry, particles ( 2 ) can be distinguished between populations ( 8 ) by combining n-dimensional parameter data, which may be derived from signal data from a particle, to mathematically achieve numerical results representative of an alteration ( 48 ). An alteration may include a rotational alteration, a scaled alteration, or perhaps even a translational alteration. Alterations may enhance separation of data points which may provide real-time classification ( 49 ) of signal data corresponding to individual particles into one of at least two populations.
1. A method comprising:
detecting with at least one detector fluorescence emitted from sperm cells in a population having X-chromosome-bearing sperm and of Y-chromosome-bearing sperm;
generating at least two signals with the at least one detector;
forming an n-dimensional parameter data for each sperm from the at least two signals, the n-dimensions including individual dimensions corresponding to respective signals in the at least two signals, wherein groupings of the X-chromosome-bearing sperm and the Y-chromosome-bearing sperm overlap in at least one of the n-dimensions;
mathematically altering the n-dimensional parameter data by at least rotating the n-dimensional parameter data to increase spatial separation in the at least one dimension having the overlap;
classifying sperm as X-chromosome-bearing sperm or Y-chromosome-bearing sperm based on the rotated n-dimensional parameter data;
generating a graphical representation of the rotated n-dimensional parameter;
discriminating between individual sperm cells in the sperm cell population based on their classification.
2. The method of claim 1 , further comprising: mathematically altering the n-dimensional parameter data by at least scaling the n-dimensional parameter data; and classifying the n-dimensional parameter data of each of the individual sperm cells as X-chromosome-bearing sperm or Y-chromosome-bearing sperm based on at least the scaled and rotated altered parameter data.
3. The method of claim 2 , further comprising: simultaneously processing the n-dimensional parameter data to alter the data by both scaling and rotation.
4. The method of claim 1 , further comprising: mathematically altering the n-dimensional parameter data by at least translating the n-dimensional parameter data; and classifying the n-dimensional parameter data of each of the individual sperm cells as X-chromosome-bearing sperm or Y-chromosome-bearing sperm based on at least the translated and rotated n-dimensional parameter data.
5. The method of claim 1 , further comprising: simultaneously processing the n-dimensional parameter data to mathematically achieve numerical results of the rotational alteration in addition to a scaled alteration and a translation alteration.
6. The method of claim 1 , wherein the step of generating a graphical representation of the at least two populations of the rotated n-dimensional parameter data comprises plotting the rotated n-dimensional parameter data in a Cartesian coordinate system.
7. The method of claim 6 , wherein plotting the rotated n-dimensional parameter data in a Cartesian coordinate system further comprises the step of plotting of the n-dimensional parameter data such that the overlap is placed on an axis of the Cartesian coordinate system.
8. The method of claim 1 , further comprising: visually distinguishing the X-chromosome-bearing sperm and Y-chromosome-bearing sperm.
9. The method of claim 8 , wherein the step of visually distinguishing the X-chromosome-bearing sperm and Y-chromosome-bearing sperm further comprises graphically representing elliptical shaped populations of X-chromosome-bearing sperm and Y-chromosome-bearing sperm having non-orthogonal angles of inclination to at least one axis; and rotating the elliptical shaped populations orthogonal to at least one axis.
10. The method of claim 1 , wherein the n-dimensional parameter data comprises 2-dimensional parameter data.
11. The method of claim 10 , wherein the subpopulation of X-chromosome-bearing sperm and the subpopulation of Y-chromosome-bearing sperm are graphically represented as generally elliptical shapes, each one having a major axis.
12. The method of claim 11 , wherein the step of mathematically altering the n-dimensional parameter data by at least rotating the 2-dimensional parameter data to increase spatial separation in the at least one dimension having the overlap further comprises rotating the 2-dimensional parameter data such that the major axis of each of the subpopulation of X-chromosome-bearing sperm and the subpopulation of Y-chromosome-bearing sperm is rotated to an orthogonal orientation with one axis of a Cartesian coordinate system.