Method for preparing permanent slides of rare sorted cells

Methods and apparatus wherein a very small number of sorted cells are attached to specified locations on a microscope slide circumscribed by well areas of a retainer slip. The cells are deposited in the wells and during centrifugation of the slide-retainer sandwich, are contacted with and attached to the serum albumin present in the well and coated on the slide. Removal of excess fluids, optional clamping means and the retainer slip followed by air drying provides for permanent attachment of rare, sorted cells in a localized, known area for subsequent staining, examination and analysis.

FIELD OF THE INVENTION 
This invention relates to a method for preparing permanent slides of cells 
sorted by electrostatic cell sorters or bulk gradient separation 
processes. 
BACKGROUND OF THE INVENTION 
Flow cytometry is a relatively new and rapidly changing technology and has 
evolved into a sophisticated analytic tool for rapidly quantitating 
multiple chemical and physical properties of individual cells or cellular 
constituents of heterogeneous populations. The value of flow cytometry in 
biological research can be seen in its ability to reveal information 
concerning the cell cycle kinetics, DNA ploidy levels, and the 
quantitation of cell surface antibodies. The sophisticated technical 
achievements made possible by this instrumentation find clinical 
applications in cancer cell detection, blood cell counting, performing 
differential white blood cell counts, drug effectiveness studies including 
cancer chemotherapy, monitoring leukemia in other solid tumors, as well as 
uses in immunology and virology. Additional biologic applications are 
continuously being discovered even as the flow cytometry techniques 
continue to undergo dramatic improvements. 
Typically, cell sorting is accomplished by instruments generally measuring 
a multitude of parameters. Most conventional cell cytometers employ some 
type of hydrodynamic focusing whereby the cells to be measured are aligned 
in a single file fashion within a fluid sheath. The cells are then passed 
by a detector region which measure either a change in electrical 
properties of a small aperture as the cell passes therethrough or the 
light scattering effects occasioned by the passage of the cell past a 
light source. Since different cells typically exhibit varying 
characteristics, detection of the dynamic effects occasioned by the 
passage of the cell provides data useful in discriminating and 
quantitating between cell populations. With the addition of cell sorting 
capabilities, such an instrument can analyze a mixed population of cells, 
discriminate between cell types and physically manipulate the cells, 
generally by employing electrostatic principles or variations thereof, so 
that they may be concentrated to greater purity. This is generally 
accomplished by the deposition of the cells into vials or similar type of 
containers. 
In order to monitor the type of cells collected in this fashion and to 
appropriately adjust the window parameters for the effective 
discrimination for and collection of desired cell types, the cells must be 
examined microscopically. With large numbers of cells, this may be 
accomplished by the deposition of the cells onto a microscope slide, and 
after staining procedures, easily found because of their pervasive 
presence on the slide. Great difficulty is encountered, however, when the 
population of selected cells is very small, i.e. on the order of a hundred 
cells. Indiscriminate application of such a small number of cells to a 
microscope slide will force the microscopist on a hunt and search detail 
in order to assure that he has correctly identified representative cell 
types in that small population. 
In order to reduce this difficult and demanding task, conventional methods 
have dictated the use of microscope slides having concave areas for the 
deposition of cells in one spot. This method has been effectively employed 
in tissue culture and tissue typing since the accumulation of cells in a 
localized spot has aided in the detection of seralogical reactions. For 
examination of single cells, this method is not desirable since all the 
cells are coalesced in a very localized area and consequently, often 
physically overlap one another. Further, such a system is primarily useful 
for cells in suspension and does not allow the facile employment of 
typical staining methods such as Wright stain and the like. 
It is an object of the present invention to provide an apparatus capable of 
permitting the examination of a small number of cells that have been 
sorted by a cell sorter and to provide their attachment to a microscope 
slide in a manner consistent with standard staining techniques. 
Another conventional method employs a cup for the collection and retention 
of cells in conjunction with the use of a centrifuge. This system has also 
proven to be ineffective since the cups are designed to contain a large 
volume and the desired rare cells are typically in very small numbers. 
With such a high dilution, it consequently becomes extraordinarily 
difficult to locate the cells for subsequent staining and examination. 
It is an object of the present invention to provide apparatus and 
methodology whereby rare sorted cells may be permanently attached onto a 
slide for subsequent staining and examination and that despite the small 
number of cells, their fixation, staining, and localization in a specified 
limited site provides for reduced probability of loss and destruction as 
well as increased handling ease. 
SUMMARY OF THE INVENTION 
In accordance with the principles and objectives of the invention, there is 
provided a system for collecting and preparing permanent slides of rare 
sorted cells comprising means for retaining, in specified locations, 
sorted cells having wells and adapted for contacting a microscope slide to 
form a slide-retainer sandwich. Prior to forming the slide-retainer 
sandwich, the sides of the microscope slide and retainer means are coated 
with serum albumin which aids in the elimination of capillary seepage and 
retention of the cells. An additional amount of serum albumin is added to 
the wells and the sorted cells are then added thereto. Centrifugation of 
the slide-retainer sandwich results in the attachment of the cells to the 
microscope slide via the serum albumin. Thereafter, the retainer means and 
excess fluids are removed and the cells, attached to the slide in known, 
localized areas, are ready for subsequent staining. 
BRIEF DESCRIPTION OF THE DRAWINGS 
The objectives of the invention and the preferred embodiments thereof will 
best be understood by reference to the accompanying drawings wherein:

BEST MODE FOR CARRYING OUT THE INVENTION 
FIG. 1 illustrates the manner in which the device of the present invention 
is to be employed. A standard microscope slide 1 is cleaned using ethanol 
or alcohol wipes preferably containing approximately seventy percent 
isopropyl alcohol. Although the slide shown has a frosted area intended 
for labeling purposes, such an area is an optional feature to be selected 
at the whim of the user and is not required for the operation of the 
invention. The cell retaining cover slip or retaining means 2 is 
preferably dimensioned for contact with the working area of the microscope 
slide 1. The retainer 2 will have at least one well 3 but, preferably will 
have a plurality of wells in order to allow for multiple sample analysis. 
The diameter, spacing and number of wells 3 will be adjusted in accordance 
with sample volumes and cell numbers contained therein and user 
preference. Well diameters of 0.170 inches and spacing between centers of 
1/2 an inch have been found effective. 
Prior to contacting the retainer cover slip 2 to slide 1, surfaces 4 and 5 
of the retainer slip and slide respectively are coated with an effective 
layer of serum albumin. the serium albumin is preferably bovine serum 
albumin in a relatively low concentration such as 2-5% in phosphate 
buffered saline. The diluted bovine serum albumin solution will preferably 
contain sodium azide at a concentration in the neighborhood of 0.01% as a 
bacteriostat. Application of the bovine serum albumin can be effectively 
accomplished by smearing one surface with the edge of another microscope 
slide similar to the technique typically employed in making blood sample 
smears. 
The retainer slip 2 is then contacted with the slide 1 to form a sandwich 
12 as shown in FIG. 2. In order to assist in maintaining the integrity of 
the sandwich, it is preferred that a retaining clamp 6 is added. If a 
clamp is employed such a feature although preferred, is optional; the 
wells 3 on retainer clip 2 are advantageously located appropriately in 
order to avoid conflicting contact. 
With reference to FIG. 2, additional serum albumin is added to well 3 to 
form a built up layer 13 contained by the well. Preferably, the same 
concentration and type of serum albumin is employed and a sufficient 
volume is used to fill the well approximately 1/3-1/2 full. Using a 
retaining cover slip having a thickness of 0.060 inches and a well having 
a diameter of 0.170 inches, it has been found that an effective volume is 
approximately 6 microliters. Thereafter, the cells are sorted either 
directly into the well from the cell sorter apparatus or are added via 
micropipette 14 or other means. It is preferable that the total volume 
added to the well 3 be limited to approximately 8 microliters in order to 
avoid spillage and loss of cells. The slide-retainer sandwich with the 
cells is then placed in a Cytocentrifuge.TM. produced by Shandon or its 
equivalent. The well opening faces the center of the rotor carousel so 
that the centrifugal force is applied to the sandwich 12 as shown in FIG. 
3. The sandwich is then centrifuged at approximately 600 rpm for about 4 
minutes so that the cells are contacted with and retained by the serum 
albumin. The figures for rotational speed and time of centrifuging may be 
adjusted as necessary in order to effect firm attachment of the cells and 
avoid morphological damage. 
The excess supenatant from the original cell suspension is then preferably 
removed at this time by siphoning off the fluid using filter paper, 
micropipette or porous plastic means as illustrated by the tissue 
representation 15 in FIG. 2. Separate siphoning means are preferably 
employed for each well in order to prevent contamination. Proper removal 
of the supernatant reduces the possibility of cell membrane destruction 
upon evaporation of surrounding saline. 
At this point, the cell retaining slip 2 is removed from the slide 1 in a 
manner avoiding disturbance of the cell areas. Preferably this is done by 
grasping the microscope slide 1 at the frosted area and holding the 
sandwich at a slight angle. The retainer cover slip is then slowly and 
gently removed by lifting the small protruding handles 11 shown in FIG. 4. 
It is preferred that as this physical operation is accomplished, a line of 
serum albumin solution should be seen to recede towards the end of the 
slide without passing through the area where the cells are secured. 
Remaining excess serum albumin may be removed with a piece of tissue or 
lense paper, again avoiding those areas where the cells have been 
attached. The slide and the attached cells are then allowed to dry 
preferably by simple evaporation. The cells may then be conveniently 
stained as desired.