Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
1999-07-02
2002-04-16
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C422S073000, C209S003200, C209S004000, C209S127400, C209S552000, C209S576000, C209S906000
Reexamination Certificate
active
06372506
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus and method for verifying the drop delay in a flow cytometer. More particularly, the present invention relates to an apparatus and method which detects the presence or absence of particles of interest in fluid droplets formed by a flow cytometer to determine whether the time at which the fluid droplets are being charged is correct, so that the droplets can be sorted electrostatically with precision.
Flow cytometers known in the art are used for analyzing and sorting particles in a fluid sample, such as cells of a blood sample or particles of interest in any other type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer transports the particles (hereinafter called “cells”) in the fluid sample as a cell stream to a flow cell, while also directing the sheath fluid to the flow cell.
Within the flow cell, a liquid sheath is formed around the cell stream to impart a substantially uniform velocity on the cell stream. The flow cell hydrodynamically focuses the cells within the stream to pass through the center of a laser beam. The point at which the cells intersect the laser beam, commonly known as the interrogation point, can be inside or outside the flow cell. As a cell moves through the interrogation point, it causes the laser light to scatter. The laser light also excites components in the cell stream that have fluorescent properties, such as fluorescent markers that have been added to the fluid sample and adhered to certain cells of interest, or fluorescent beads mixed into the stream.
The flow cytometer further includes an appropriate detection system consisting of photomultiplier tubes, photodiodes or other light detecting devices, which are focused at the intersection point. The flow cytometer analyzes the detected light to measure physical and fluorescent properties of the cell. The flow cytometer can further sort the cells based on these measured properties.
To sort cells by an electrostatic method, the desired cell must be contained within an electrically charged droplet. To produce droplets, the flow cell is rapidly vibrated by an acoustic device, such as a piezoelectric element. The droplets form after the cell stream exits the flow cell and at a distance downstream from the interrogation point. Hence, a time delay exists from when the cell is at the interrogation point until the cell reaches the actual break-off point of the droplet. The magnitude of the time delay is a function of the manner in which the flow cell is vibrated to produce the droplets, and generally can be manually adjusted, if necessary.
To charge the droplet, the flow cell includes a charging element whose electrical potential can be rapidly changed. Due to the time delay which occurs while the cell travels from the interrogation point to the droplet break-off point, the flow cytometer must invoke a delay period between when the cell is detected to when the electrical potential is applied to the charging element. This charging delay is commonly referred to as the “drop delay”, and should coincide with the travel time delay for the cell between the interrogation point and the droplet break-off point to insure that the cell of interest is in the droplet being charged.
At the instant the desired cell is in the droplet just breaking away from the cell stream, the charging element is brought up to the appropriate potential, thereby causing the droplet to isolate the charge once it is broken off from the stream. The electrostatic potential from the charging circuit cycles between different potentials to appropriately charge each droplet as it is broken off from the cell stream.
Because the cell stream exits the flow cell in a substantially downward vertical direction, the droplets also propagate in that direction after they are formed. To sort the charged droplet containing the desired cell, the flow cytometer includes two or more deflection plates held at a constant electrical potential difference. The deflection plates form an electrostatic field which deflects the trajectory of charged droplets from that of uncharged droplets as they pass through the electrostatic field. Positively charged droplets are attracted by the negative plate and repelled by the positive plate, while negatively charged droplets are attracted to the positive plate and repelled by the negative plate. The lengths of the deflection plates are small enough so that the droplets which are traveling at high velocity clear the electrostatic field before striking the plates. Accordingly, the droplets and the cells contained therein can be collected in appropriate collection vessels downstream of the plates.
Known flow cytometers similar to the type described above are described, for example, in U.S. Pat. Nos. 3,960,449, 4,347,935, 4,667,830, 5,464,581, 5,483,469, 5,602,039, 5,643,796 and 5,700,692, the entire contents of each patent being incorporated by reference herein. Other types of known flow cytometer, are the FACSVantage™, FACSort™, FACSCount™, FACScan™ and FACSCalibur™ systems, each manufactured by Becton Dickinson and Company, the assignee of the present invention.
As can be appreciated from the foreign description, in order for a flow cytometer to correctly sort cells of interest, the drop delay must be precisely measured to ensure that a cell of interest which was detected at the interrogation point is actually present in the droplet being charged. If the drop delay is not accurately determined, it is likely that the charge will be applied to a droplet formed earlier or later than the droplet containing the cell of interest. In this event, the droplet containing the cell of interest will not be charged, and therefore will not be sorted as desired. Rather, an incorrectly charged droplet will be sorted, thus reducing the overall sorted cell count, or adding an unwanted cell to the cell count if that droplet contains an unwanted cell.
Several known methods exist for calculating the drop delay with reasonable accuracy. In one known method, the distance between the interrogation point and the droplet formation (break-off) point is measured using, for example, a graduated optical measuring tool. The measuring tool is then repositioned so that the graduation originally positioned at the interrogation point is moved to the droplet break-off point, and the graduation originally positioned at the droplet break-off point is positioned in the droplet stream. The number of droplets appearing between the graduation positioned at the droplet break-off point and the graduation in the droplet stream is then counted, and the drop delay is expressed as the number of counted drops.
For example, if the number of counted drops appearing between the graduations is equal to five, this indicates that five drop periods elapse form the time a cell is at the interrogation point until it reaches the droplet break-off point. Accordingly, the charge timing of the flow cytometer is set so that charging intended to be applied to a droplet containing a cell of interest is delayed by five drop periods from the time when the cell of interest is detected at the interrogation point.
Although this method generally enables the flow cytometer to charge the correct droplets, and therefore sort the cells of interest with reasonable accuracy, the method provides no means to verify the accuracy of the charge timing while cell sorting is being performed. Rather, the results of the sort must be examined after all or at least a portion of the sorting process has been completed. If, upon examination of the results, it is determined that the charge timing was incorrect, the process must be repeated until the correct charge timing is determined. Furthermore, because the method requires multiple steps, such as aligning the graduations on the optical instrument with the appropriate points at different positions along the droplet stream, the process can
Becton Dickinson and Company
Fiedler Alan W.
Wallenhorst Maureen M.
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