Method for preserving cells, and uses of said method

This invention provides a method for preserving cells which comprises the steps of (a) suspending cells in a physiologically-acceptable, isotonic medium; and (b) fixing the cells so suspended at a temperature of less than about 10.degree. C. under sufficiently hypertonic conditions so as to disperse the cells in a single, unagglutinated state, thereby preserving cells. This invention also provides a method for detecting cells separated from a sample which have been preserved according to the aforementioned method. This invention also provides a method for visualizing cells. Also provided is a method for detecting a metabolic process in cells present in a sample. This invention also provides a method for detecting the presence of rare cells in a sample which specifically possess on their surfaces a moiety recognized by a known ligand comprising preserving cells separated from the sample according to the aforementioned method for preserving cells. The subject invention also provides for quantitatively determining and isolating rare cells detected according to the aformentioned method. Further provided are methods for determining whether a fetus is afflicted with a genetic abnormality, determining the gender of a fetus, determining whether a subject is afflicted with a tumor, and detecting in a subject the presence of cells expressing a malignant phenotype, each method comprising preserving cells according to the aforementioned method for preserving cells.

Throughout this application, various references are referred to within 
parentheses. Disclosures of these publications in their entireties are 
hereby incorporated by reference into this application to more fully 
describe the state of the art to which this invention pertains. Full 
bibliographic citation for these references may be found at the end of 
this application, preceding the claims. 
BACKGROUND OF THE INVENTION 
Rare cells are often circulating in peripheral blood (1-5). For example, 
the frequency of circulating trophoblasts obtained from a subject carrying 
a 47, XYY fetus was approximately one in 10.sup.5 nucleated blood cells 
after pre-enrichment by gradient fractionation (5). The percentage of 
mononuclear cells infected with Human Immunodeficiency Virus (HIV) in the 
peripheral blood of HIV-infected subjects was 0.003-0.01% in asymptomatic 
subjects and 1-5% in subjects displaying symptoms associated with advanced 
stages of the disease (6-9). However, a technique for preserving cells 
obtained from biological specimens so that rare cells can be analyzed or 
isolated at a later time has not heretofore been disclosed. 
Rare cells are a potential source for early detection or diagnosis of 
infection, malignancy, or genetic disorder. A technique for preserving 
cells obtained from biological specimens in a single, unagglutinated state 
would provide a means for such sensitive diagnosis. A single sample 
obtained from a subject containing cells which are preserved in a single, 
unagglutinated state may be analyzed multiple times, varying the 
parameters of analysis each time as desired yet avoiding the necessity of 
repeatedly obtaining samples from the same subject. Furthermore, preserved 
cells from different subjects may be more efficiently analyzed than cells 
from different subjects maintained in a viable state. Cells from a single 
subject obtained at different times or stages during the course of a 
disease may likewise be analyzed in a single, unagglutinated state at one 
time. 
SUMMARY OF THE INVENTION 
This invention provides a method for preserving cells which comprises the 
steps of (a) suspending cells in a physiologically-acceptable, isotonic 
medium; and (b) fixing the cells so suspended at a temperature of less 
than about 10.degree. C. under sufficiently hypertonic conditions so as to 
disperse the cells in a single, unagglutinated state, thereby preserving 
the cells. 
This invention also provides a method for detecting cells present in a 
sample which comprises the steps of (a) separating cells from the sample; 
(b) preserving the cells so separated according to the above-described 
method; and (c) detecting cells preserved in step (b), so as to thereby 
detect cells present in the sample. 
This invention also provides a method for visualizing cells present in a 
sample which comprises the steps of (a) separating cells from the sample; 
(b) preserving the cells so separated according to the above-described 
method; and (c) visualizing the cells preserved in step (b) so as to 
thereby visualize cells present in the sample. 
This invention also provides a method for detecting a metabolic process in 
cells present in a sample which comprises the steps of (a) separating 
cells from the sample; (b) contacting the cells so separated with a 
detectable substance capable of interacting with a metabolic substance in 
the pathway of the metabolic process; (c) collecting cells resulting from 
step (b) at a plurality of suitably spaced points in time; (d) preserving 
the cells so collected at each time point according to the above-described 
method of preserving cells; (e) detecting the presence of the detectable 
substance in the cells so preserved at each time point; and (f) 
determining the existence of a change in the presence of the detectable 
substance so detected, the existence of the change known to result from 
the metabolic process, so as to thereby detect the metabolic process in 
the cells present in the sample. 
This invention also provides a method for detecting the presence of rare 
cells in a sample which cells specifically possess on their surfaces a 
moiety recognized by a known ligand which comprises the steps of (a) 
separating cells from the sample; (b) contacting the cells so separated 
with a known ligand, said ligand being detectable, under conditions which 
would permit the known ligand to specifically form a complex with the 
moiety recognized thereby, so as to thereby label cells possessing the 
moiety on their surfaces if present in the sample; (c) removing remaining 
uncomplexed ligand; (d) preserving the resulting cells according to the 
method of preserving cells described above so as to thereby preserve any 
cells labeled in step (b); and (e) detecting the presence of any labelled 
cells so preserved so as to thereby detect the presence of rare cells in 
the sample which specifically possess on their surfaces the moiety 
recognized by the known ligand. 
Also provided is a method which comprises quantitatively determining cells 
detected according to the above-described method for detecting rare cells 
present in a sample, as well as a method which comprises isolating cells 
detected according to the above-described method. 
This invention further provides a method for determining whether a fetus is 
afflicted with a genetic abnormality which comprises the steps of (a) 
obtaining a sample comprising fetal cells from the fetus, which sample 
comprises fetal cells which specifically possess on their surfaces a 
moiety recognized by a known ligand; (b) detecting the presence of fetal 
cells in the sample so obtained according to the above-described method 
for detecting rare cells present in a sample; and (c) determining whether 
the fetal cells so detected are characteristic of cells from a fetus 
having the genetic abnormality, so as to thereby determine whether the 
fetus is afflicted with the genetic abnormality. 
This invention also provides a method for determining the gender of a fetus 
which comprises the steps of (a) obtaining a sample comprising fetal cells 
from the fetus, which sample comprises fetal cells which specifically 
possess on their surfaces a moiety recognized by a known ligand; (b) 
detecting the presence of fetal cells in the sample so obtained according 
to the above-described method for detecting the presence of rare cells in 
a sample; and (c) determining whether the fetal cells so detected possess 
two X chromosomes or an X chromosome and a Y chromosome, so as to thereby 
determine the gender of the fetus. 
Also provided by the subject invention is a method of determining whether a 
subject is afflicted with a tumor normally tending to shed cells into the 
blood of a subject afflicted therewith which comprises the steps of (a) 
obtaining a blood sample from the subject; (b) removing erythrocytes from 
the blood sample so obtained; and (c) detecting the presence of tumor 
cells in the resulting sample according to the above-described method for 
detecting the presence of rare cells in a sample, so as to thereby 
determine whether the subject is afflicted with said tumor. 
This invention further provides a method of detecting in a subject the 
presence of cells expressing a malignant phenotype which comprises the 
steps of (a) obtaining a suitable sample from the subject; (b) removing 
erythrocytes from the sample so obtained; and (c) detecting the presence 
of cells expressing the malignant phenotype in the resulting sample 
according to the above-described method of detecting the presence of rare 
cells in a sample, so as to thereby detect in the subject the presence of 
cells expressing the malignant phenotype. 
This invention also provides a method for determining whether a subject who 
has been treated with an anti-tumor therapy, said therapy having included 
chemotherapy, possesses cells expressing a malignant phenotype which have 
undergone a genetic alteration associated with drug resistance, which 
comprises the steps of (a) detecting in the subject the presence of cells 
expressing a malignant phenotype according to the above-described method 
and; (b) determining whether any cells so detected are characteristic of 
cells which have undergone a genetic alteration associated with drug 
resistance, thereby determining whether the subject possesses cells 
expressing a malignant phenotype which have undergone a genetic alteration 
associated with drug resistance. 
DETAILED DESCRIPTION OF THE INVENTION 
This invention provides a method for preserving cells which comprises the 
steps of (a) suspending cells in a physiologically-acceptable, isotonic 
medium; and (b) fixing the cells so suspended at a temperature of less 
than about 10.degree. C. under sufficiently hypertonic conditions so as to 
disperse the cells in a single, unagglutinated state, thereby preserving 
the cells. 
The method of the subject invention may further comprise a step of 
separating cells from a cell-containing sample. Examples of 
cell-containing samples are well-known in the art and include, but are not 
limited to, biological specimens such as biopsies, tissue aspirates, 
lavages, blood samples including both specific and peripheral blood 
samples, semen, and urine. Suitable conditions for separating cells from a 
cell-containing sample are any conditions or procedures known to those of 
ordinary skill in the art for removing all or most of the extracellular 
material in a cell-containing sample from the cells in the sample. Such 
procedures are well-known in the art and include, but are not limited to, 
grinding and sieving to remove particulate matter from a sample, digesting 
any extracellular matrix in a sample, and centrifuging to remove 
extracellular fluid from a sample. Preferably, the cells are separated 
from the cell-containing sample at a temperature of from about 0.degree. 
C. to about 10.degree. C. 
If the cells separated from a sample include erythrocytes, unless 
preservation of the erythrocytes is desired, the erythrocytes are 
preferably removed after separation of the cells from the extracellular 
material. Any method for removing erythrocytes known in the art may be 
used in the subject invention, so long as the cells to be preserved remain 
viable until they are contacted with the fixative used for fixing the 
cells. For example, the erythrocytes may be removed by centrifugation 
through a density gradient. Alternatively, the erythrocytes may be lysed 
and the nucleated cells recovered after centrifugation. Methods for lysing 
erythrocytes are known to those of ordinary skill in the art and include 
contacting the erythrocytes with a known lysing reagent. If a known lysing 
reagent is used, the lysing reagent is preferably attenuated with serum so 
as to be physiologically compatible with the nucleated cells to be 
preserved. Also preferred as a lysing reagent is ammonium chloride. In one 
embodiment, the lysis occurs at a temperature of from about 0.degree. C. 
to about 10.degree. C. In another embodiment, the lysis occurs at a 
temperature of from 0.degree. C. to about 4.degree. C. In the preferred 
embodiment, the lysis occurs at a temperature of about 4.degree. C. 
As used herein, the term "physiologically-acceptable, isotonic medium" 
means any medium in which cells remain viable. Such media are well known 
to those of ordinary skill in the art and include phosphate-based buffers; 
bicarbonate-based buffers; and citrate-based buffers. For certain 
applications of the method of the subject invention, preservation of the 
protein moieties of the cells may be especially desired, for example where 
antigens on the surfaces of the cells are to be used for later 
immunodetection of the preserved cells. For such applications, a 
phosphate-based buffer is preferred as the physiologically-acceptable, 
isotonic medium. For other applications of the method of the subject 
invention, preservation of the nucleic acid moieties of the cells may be 
especially desired, for example where the DNA in the cells is to be 
hybridized with a nucleic acid probe. For these applications, a 
citrate-based buffer is preferred as the physiologically-acceptable, 
isotonic medium. An isotonic medium is any medium which contains a 
concentration of solutes substantially identical to the concentration of 
solutes inside a cell, i.e. a medium of about 0.3 molarity. 
The pH of physiologically-acceptable media are any pH in which cells remain 
viable. pH at which cells remain viable are known to those of ordinary 
skill in the art and are typically between about 5.5 and about 8.0. A pH 
of about 7.4 is preferred. 
In one embodiment, the cells are suspended in the 
physiologically-acceptable, isotonic medium at a temperature of from about 
0.degree. C. to about 10.degree. C. In another embodiment, the cells are 
suspended at a temperature of from about 0.degree. C. to about 4.degree. 
C.. In another embodiment, the cells are suspended at a temperature of 
about 4.degree. C. 
After the cells are suspended in the physiologically-acceptable, isotonic 
medium, they are fixed at a suitable temperature and under sufficiently 
hypertonic conditions so as to disperse the cells in a single, 
unagglutinated state. In the subject invention, the cells may be fixed by 
contacting them with a fixative for a sufficient period of time. As used 
herein, the term "fixing" means treating cells so as to preserve some 
structural aspect of the cells, for example the size, organization, 
protein moieties, and/or nucleic acid moieties of the cells. 
By fixing cells at a suitable temperature and under sufficiently hypertonic 
condition so as to disperse the cells in a single, unagglutinated state, 
the individual cells may be analyzed or observed without obstruction from 
one another either immediately or many months or even years after they 
have been preserved. 
Any fixative may be used in the above described method, provided the 
fixative fixes the cells gradually enough so that the cells are fixed 
while the temperature and hypertonicity of the suspension are such that 
the cells are dispersed in a single, unagglutinated state. Preferably, the 
fixative is formaldehyde. 
If the fixative is formaldehyde, the final concentration of formaldehyde in 
the solution in which the cells are fixed should be such that the cells 
are fixed at a suitable temperature and while the solution is sufficiently 
hypertonic so that the cells are dispersed in a single, unagglutinated 
state. Preferably, the final formaldehyde concentration is between 0.5% 
and 2%. Therefore, the concentration of formaldehyde groups in the 
solution contacting the cells should be sufficient to achieve a final 
formaldehyde concentration in the solution in which the cells are fixed 
within the aforementioned range. Formaldehyde solutions useful in the 
method of the subject invention and having a suitable aldehyde 
concentration are readily available to those of ordinary skill in the art. 
For example, formalin (approximately 37% formaldehyde in methanol) may be 
used to fix the cells in the method of the subject invention. 
Alternatively, paraformaldehyde may be dissolved to form a solution of 
short chain polymers and monomers of formaldehyde, for example by heating 
paraformaldehyde in an appropriate solvent and/or by increasing the pH of 
paraformaldehyde dispersed in an appropriate solvent. Appropriate solvents 
for dissolving formaldehyde are well known to those of ordinary skill in 
the art. The concentration of formaldehyde in the formaldehyde solution to 
be added to the cell suspension may be ascertained by means well known in 
the art, for example by adding Schiff's reagent to the formaldehyde 
solution and spectrophotometrically determining the aldehyde concentration 
as a function of the color of the solution resulting therefrom. 
For purposes of this invention, the temperature at which the cells are 
fixed is less than about 10.degree. C.. In one embodiment, the temperature 
is from about 0.degree. C. to about 10.degree. C. In another embodiment, 
the temperature is from about 0.degree. C. to about 4.degree. C. In the 
preferred embodiment, the temperature is about 4.degree. C. Without 
limiting the scope of the subject invention, it is hypothesized that such 
temperatures help disperse cells in a single, unagglutinated state by 
preventing the polymerization of microtubules in the cells and thereby 
causing the cells in the sample to "round-up", i.e. separate from one 
another. 
In the method of the subject invention, the cells are fixed under 
sufficiently hypertonic conditions such that, given the particular 
temperature of the suspension, the cells are dispersed in a single, 
unagglutinated state. As used herein, the term "hypertonic" means having a 
concentration of solutes in a solution which concentration is greater than 
the concentration of solutes inside a cell, i.e. a concentration greater 
than about 0.3 molarity. By suspending the cells in a hypertonic solution, 
the cells are separated from one another, thus helping to prevent the 
cells from agglutinating. Any solutes for making a solution hypertonic may 
be used in the subject invention. For example glucose or sucrose may be 
dissolved in the solution in which the cells are fixed. In one embodiment, 
the solution in which the cells are fixed is rendered hypertonic solely 
via the dissolved fixative, for example via the dissolved formaldehyde in 
a formaldehyde solution. A final concentration of formaldehyde in the 
isotonic medium in which the cells are suspended of between about 0.5% to 
about 2% is adequate to render the solution hypertonic to preserve cells 
in a single, unagglutinated state when the temperature is less than about 
10.degree. C. 
Optionally, chemicals may be added to the fixative prior to contacting the 
cells with the fixative in order to render the cells permeable to ligands 
which bind to intracellular moieties. Binding of ligands to intracellular 
moieties may be desired for purposes of visualizing, detecting, or 
isolating the cells after they have been preserved. Chemicals which 
increase the permeability of cells are well known in the art. The organic 
solvent acetone is preferred as a chemical for increasing the permeability 
of cells when preservation of protein moieties is desired. The organic 
solvent methanol is preferred as a chemical for increasing the 
permeability of cells when preservation of nucleic acid moieties is 
desired. The organic solvent ethanol is another chemical known in the art 
to increase the permeability of cells. 
Alternatively, the cells may be suspended in a solution of formaldehyde 
combined with lysine, sucrose, and periodate for fixation of both protein 
moieties and nucleic acid moieties, or when the cells are to be studied by 
electron microscopy. A detergent may then be added to the solution in 
which the cells are fixed to permeabilize the plasma membrane and the 
nuclear envelope of the cells. 
Preferably, when chemicals which are organic solvents are added to 
permeabilize the cells, they are added so that their combined final 
concentration in the solution in which the cells are fixed is between 
about 0.3% and about 60%. The greater the final concentration of organic 
solvent in the cell suspension, the greater the permeability obtained. 
The time elapsing before the cells are fixed once the fixative has been 
added in the method of the subject invention depends on the final 
concentration of the fixative in the cell suspension in which the cells 
are fixed. The greater the final fixative concentration in the cell 
suspension, the shorter the time elapsing before the cells in the 
suspension are fixed. For example, if the cell suspension contains a final 
formaldehyde concentration of about 1%, the cells in the suspension are 
fixed in approximately thirty minutes. The temperature and the 
hypertonicity of the suspension must remain sufficient to maintain the 
cells dispersed in a single, unagglutinated state until the cells are 
fixed. However, once the cells are fixed, they may be stored indefinitely. 
This invention also provides a method for detecting cells present in a 
sample which comprises the steps of (a) separating cells from the sample; 
(b) preserving the cells so separated according to the above-described 
method; and (c) detecting cells preserved in step (b), so as to thereby 
detect cells present in the sample. 
Methods for separating cells from a sample are well-known in the art. Any 
method for separating cells can be used in the subject invention, 
including, but not limited to, the well-known methods for separating cells 
from a cell-containing sample discussed above. 
In one embodiment, all of the cells preserved in step (b) are detected. In 
another embodiment, only cells possessing a unique phenotype preserved in 
step (b) are detected. 
Methods for detecting cells are known in the art and any such method may be 
used in the method for detecting cells of the subject invention. Examples 
of methods known in the art for detecting cells include, but are not 
limited to, labelling cells with a fluorescent marker followed by analysis 
with a fluorescent activated cell sorter, and visualizing cells, for 
example as in histochemistry. 
As used herein, detecting cells includes detecting any cells which remain 
after excluding cells possessing a unique phenotype. Cells possessing a 
unique phenotype may be excluded by contacting them with a detectable 
ligand which recognizes a moiety which the cells possessing the unique 
phenotype specifically express, thereby labelling cells possessing the 
unique phenotype, and subsequently excluding any cells so labelled. 
Preservation of cells in a singe, unagglutinated state according to the 
method of the subject invention offers advantages for detecting cells. For 
example, fluorescently-labelled cells in a single, unagglutinated state 
may be more accurately detected by a fluorescence activated cell sorter 
than fluorescently-labelled cells which are partially agglutinated. 
When the method for detecting cells is visualizing the cells, any method 
known in the art for visualizing cells may be used. Examples of methods 
known in the art for visualizing cells include, but are not limited to, 
light microscopy and phase-contrast microscopy. 
Known visualization methods which may be employed in the subject invention 
may comprise staining cellular molecules and organelles with known 
staining reagents, such as eosin or methylene blue. Also, cellular 
molecules or organelles of interest may be visualized by tagging them with 
a visualizable marker such as a fluorescently labelled antibody or 
fluorescently labelled probe, as for example in fluorescence in situ 
hybridization. In the method of the subject invention, the tagging may 
occur either before or after the preservation step. Furthermore, the 
molecules or organelles may be either on the surface of the cells or in 
the interior of the cells. 
The subject invention also provides a method for visualizing cells present 
in a sample which comprises the steps of (a) separating cells from the 
sample; (b) preserving the cells so separated according to the method of 
the subject invention; and (c) visualizing the cells preserved in step (b) 
so as to thereby visualize cells present in the sample. 
Methods for visualizing cells are well-known in the art, and any such 
method may be used for visualizing the cells preserved in the method of 
the subject invention. Methods known in the art for visualizing cells are 
described above. 
As discussed above, the method of the subject invention preserves the cells 
in a single, unagglutinated state. Therefore, the method of the subject 
invention offers a means for measuring or visualizing a detectable 
substance in cells at any particular point in time, the presence of which 
substance in the cells may change over time as a result of a cellular 
metabolic process. Accordingly, this invention also provides a method for 
detecting a metabolic process in cells present in a sample which comprises 
the steps of (a) separating cells from the sample; (b) contacting the 
cells so separated with a detectable substance capable of interacting with 
a metabolic substance in the pathway of the metabolic process; (c) 
collecting cells resulting from step (b) at a plurality of suitably spaced 
points in time; (d) preserving the cells so collected at each time point 
according to the method of the subject invention; (e) detecting the 
presence of the detectable substance in the cells so preserved at each 
time point; and (f) determining the existence of a change in the presence 
of the detectable substance so detected, the existence of the change known 
to result from the metabolic process, so as to thereby detect the 
metabolic process in the cells present in the sample. 
Metabolic processes occurring in cells which may be detected according to 
the subject invention are well known in the art and include, but are not 
limited to, the metabolism of a drug in a cell or the internalization of a 
particular cell receptor. For example, the internalization of low density 
lipoprotein (LDL) receptors to which LDL has bound may be detected 
according to the subject invention. Detecting LDL receptor internalization 
would be useful, for example, for determining familial 
hypercholesterolemia. 
As used herein, a "metabolic substance" is any substance which participates 
in or is produced by a metabolic process. In one embodiment, the metabolic 
substance is a metabolite of the metabolic process. In another embodiment, 
the metabolic substance is an intermediate of the metabolic process. In 
still another embodiment, the metabolic substance is a product of the 
metabolic process. In still another embodiment, the metabolic substance is 
a byproduct of the metabolic process. 
As used herein, "the pathway of the metabolic process" indicates the series 
of interactions of those metabolic substances which make up the metabolic 
process. Such interactions include, but are not limited to, formations of 
complexes and chemical reactions. 
Substances capable of interacting with a metabolic substance in the pathway 
of a metabolic process useful in the subject invention are well-known and 
are to be selected based upon the metabolic substance, which in turn is 
selected based upon the particular metabolic process to be detected. For 
example, if the metabolic process to be detected is the metabolism of a 
drug, the metabolic substance may be an enzyme capable of converting the 
drug to a product, and the substance capable of interacting with the 
enzyme may be the drug itself. As another example, if the metabolic 
process to be detected is the internalization of a particular cell 
receptor, the metabolic substance may be the cell receptor, and the 
substance interacting with the cell receptor a ligand which binds to the 
cell receptor or an antibody which specifically recognizes the cell 
receptor. 
As used herein, substances capable of interacting with a metabolic 
substance include, but are not limited to, substances which are capable of 
forming a specific complex with the metabolic substance, as well as 
substances which are capable of reacting with the metabolic substance so 
as to form a distinct product. 
In the method for detecting a metabolic process described above, the 
substance capable of interacting with the metabolic substance in the 
pathway of the metabolic process must be detectable. Detectable substances 
are well-known to those of ordinary skill in the art and include, but are 
not limited to, substances conjugated to a detectable marker, such as a 
fluorophore; enzymes which may convert a substrate into a chromogenic 
product; and molecules which are specifically recognized by a known 
antibody. 
Changes in the presence of the detectable substance known to result from a 
particular metabolic process are ascertainable by means known to those of 
ordinary skill in the art. Such changes include, but are not limited to, 
changes in the location of the substance within the cell, or changes in 
the quantity of the substance. 
Preservation of the cells in a single, unagglutinated state according to 
the method of the subject invention offers advantages for certain 
applications, such as for detecting or isolating rare cells. Accordingly, 
in one embodiment of the method of the subject invention, the cells 
preserved include rare cells. As used herein, the term "rare cells" means 
cells possessing a unique phenotype whose frequency in a sample is no 
greater than about 5% of the total cell population in said sample. In one 
embodiment, the frequency of the cells is not greater than about 1%. In 
another embodiment, the frequency is not greater than about 0.1%. In still 
another embodiment, the frequency is less than 0.0001%. Assume, for 
example, that cells possessing phenotype X constitute only about 0.0001% 
of the total number of cells in a sample, said sample being blood. Cells 
possessing the phenotype X are therefore rare cells for purposes of the 
subject invention. 
Rare cells may be either nucleated or non-nucleated. Rare cells include, 
but are not limited to, the following cells found in certain blood 
samples: cells expressing a malignant phenotype; fetal cells, such as 
fetal cells in maternal peripheral blood; tumor cells, such as tumor cells 
which have shed from a tumor into an afflicted subject's blood; cells 
infected with a virus, such as cells infected with HIV; cells transfected 
with a gene of interest; and aberrant subtypes of T-cells or B-cells 
present in the peripheral blood of subjects afflicted with autoreactive 
disorders. 
Rare cells possessing a particular phenotype include rare cells which 
specifically possess on their surfaces a moiety recognized by a known 
ligand. The moiety recognized by the known ligand may be complexed with 
the known ligand after the cells have been preserved according to the 
method of the subject invention. Alternatively, the moiety recognized by 
the known ligand may be complexed with the known ligand prior to 
preservation of the cells according to the method of the subject 
invention. Accordingly, this invention also provides a method for 
detecting the presence of rare cells in a sample which cells specifically 
possess on their surfaces a moiety recognized by a known ligand which 
comprises the steps of (a) separating cells from the sample; (b) 
contacting the cells so separated with the known ligand, said ligand being 
detectable, under conditions which would permit the known ligand to 
specifically form a complex with the moiety recognized thereby so as to 
thereby label cells possessing the moiety on their surfaces if present in 
the sample; (c) removing remaining uncomplexed ligand; (d) preserving the 
resulting cells according to the method of the subject invention so as to 
thereby preserve any cells labelled in step (b); and (e) detecting the 
presence of any labelled cells so preserved so as to thereby detect the 
presence of rare cells in the sample which specifically possess on their 
surfaces the moiety recognized by the known ligand. 
Any method for separating cells from a sample may be used in the method 
described above, and such methods for separating cells from a sample are 
well-known in the art as described above. 
In one embodiment of the method for detecting the presence of rare cells in 
a sample which specifically possess on their surfaces a moiety recognized 
by a known ligand, the rare cells which specifically possess on their 
surfaces a moiety recognized by a known ligand are nucleated cells. In 
different embodiments when the rare cells are nucleated cells, the rare 
cells are cells expressing a malignant phenotype, fetal cells, tumor 
cells, cells infected with a virus, or cells transfected with a gene of 
interest. 
Nucleated cells which express a malignant phenotype and which specifically 
possess on their surfaces a moiety recognized by a known ligand include, 
but are not limited to, leukemia cells, the phenotype of which is the 
presence of the calla antigen on their cell surfaces. 
Ligands which form a complex with moieties on the surfaces of cells which 
express a malignant phenotype are well known and may be obtained by means 
well known to those of ordinary skill in the art. For example, a ligand 
which recognizes a moiety on the surfaces of the cells expressing the 
malignant phenotype may be obtained by known immunological methods, such 
as by injecting a mammal with cells which possess on their surfaces the 
moiety and subsequently obtaining the antiserum from the mammal, or by 
forming hybridomas from the spleen cells of a mammal infected with cells 
which possess on their surfaces the moiety and subsequently obtaining 
monoclonal antibodies which recognize the moiety from the hybridomas. An 
example of a known ligand which recognizes a moiety on the surface of a 
cell expressing a malignant phenotype is the anti-calla antibody, which 
recognizes an epitope of the calla antigen. 
Fetal cells which specifically possess on their surfaces a moiety 
recognized by a known ligand are well known in the art and include, but 
are not limited to, trophoblasts. 
Ligands which recognize moieties which fetal cells specifically possess on 
their surfaces are well known and may be obtained using methods known in 
the art, for example the well-known immunological techniques for producing 
polyclonal and monoclonal antibodies described above. Examples of known 
antibodies which bind to epitopes which fetal cells specifically possess 
on their surfaces include, but are not limited to, the anti-trophoblast 
monoclonal antibodies FD066Q (10), FD046B (10), and PI 153/3 (ATCC No. TIB 
198). 
Tumor cells which specifically possess on their surfaces a moiety 
recognized by a known ligand are well known to those of ordinary skill in 
the art. In one embodiment, these tumor cells are tumor cells originating 
from a tumor normally tending to shed cells into the blood stream of a 
subject afflicted therewith. Such tumors include, for example, melanomas, 
breast tumors, spleen tumors, liver tumors, and kidney tumors. 
Ligands which bind to moieties which tumor cells specifically possess on 
their surfaces are well known and may be obtained using methods well known 
to those of ordinary skill in the art, for example the well-known 
immunological techniques for producing antibodies described above. Ligands 
which bind to moieties which tumor cells specifically possess on their 
surfaces include, but are not limited to, the monoclonal antibodies D14 
(ATCC No. HB 8439) and M111 (ATCC No. HB 8438), each of which recognize 
epitopes on the surfaces of melanomas, and the monoclonal antibody 317G5 
(ATCC No. HB 8485) which recognizes an epitope on the surfaces of cells 
from breast tumors. 
Cells which are infected with a virus and specifically possess on their 
surfaces moieties unique to the virus and recognized by a known ligand are 
well known to those of ordinary skill in the art. Examples of cells 
infected with a virus which specifically possess on their surfaces a 
moiety recognized by a known ligand include, but are not limited to, cells 
infected with Human Immunodeficiency Virus (HIV) which, for example, 
express on their surfaces as a moiety the HIV viral envelope protein and 
therefore specifically possess epitopes on their surfaces unique to the 
HIV viral envelope protein. 
Ligands which recognize moieties which cells infected with a virus 
specifically possess on their surfaces are well known and may be obtained 
using methods well known to those of ordinary skill in the art, for 
example the immunological methods for producing antibodies discussed 
above. Examples of ligands which recognize moieties specifically on the 
surfaces of cells infected with a virus include, but are not limited to, 
the anti-gp120 antibody 0.5 .beta. (NIH AIDS Reference Reagent No. 904), 
which recognizes the HIV viral envelope protein specifically on the 
surfaces of HIV infected cells. Further examples of ligands which 
recognize moieties specifically on the surfaces of cells infected with a 
virus include the monoclonal antibody designated 0.5 .alpha. (ATCC No. HB 
8755). The 0.5 .alpha. antibody recognizes an epitope specifically present 
on the surfaces of cells infected with Human T-cell Leukemia Virus type 1 
(HTLV-1). 
Cells transfected with a gene of interest which specifically possess on 
their surfaces a moiety recognized by a known ligand are well known in the 
art. As used herein, the phrase "gene of interest" means a gene which may 
be transfected into a cell. An example of a gene of interest is a gene 
encoding a receptor, such as the T cell CD4 receptor. The subject method 
for detecting cells transfected with a gene of interest would be useful, 
for example, for detecting cells transfected with a gene encoding a 
putative receptor in a sample comprising cells transfected with a genetic 
library, by contacting the sample with a known ligand to the putative 
receptor. 
When the rare cells which specifically possess on their surfaces a moiety 
recognized by a known ligand to be detected according to the subject 
method are nucleated cells, the method of the subject invention preferably 
comprises removing any erythrocytes prior to preserving the cells. Any 
method for removing erythrocytes known to those of ordinary skill may be 
used to remove erythrocytes in the method of the subject invention. 
Methods for removing erythrocytes are well known in the art as described 
above. If a known lysing reagent is used in the process of removing the 
erythrocytes, the lysing reagent is preferably attenuated with serum so as 
to be physiologically compatible with the nucleated cells to be preserved. 
Also preferred as a lysing reagent is ammonium chloride. In one 
embodiment, the lysis occurs at a temperature of from about 0.degree. C. 
to about 10.degree. C. In another embodiment, the lysis occurs at a 
temperature of from 0.degree. C. to about 4.degree. C. In another 
embodiment, the lysis occurs at a temperature of about 4.degree. C. After 
lysis, the nucleated cells may be recovered, for example, by 
centrifugation. 
In one embodiment of the subject method for detecting rare cells which 
specifically possess on their surfaces a moiety recognized by a known 
ligand, the moiety is a receptor. In another embodiment, the moiety is an 
epitope and the known ligand is an antibody which binds to the epitope. 
Receptors and epitopes specifically on the surfaces of cells and the 
ligands recognizing them are well known in the art and include, but are 
not limited to, the receptors, epitopes and ligands described above. 
In the subject method for detecting rare cells which specifically possess 
on their surfaces a moiety recognized by a known ligand, the known ligand 
must be a detectable ligand. Detectable ligands are known to those of 
ordinary skill in the art and include, but are not limited to, 
radiolabeled substances; iron-binding substances such as ferritin; 
magnetically charged substances; ligands conjugated to a detectable 
marker, such as an enzyme capable of generating a chromogenic product, or 
a fluorescent dye such as fluorescein; or ligands permitting detection 
through contact with a second detectable ligand. Any detectable ligand 
known in the art may be used in the subject invention and may be selected 
based on the anticipated application for the preserved cells. If the 
detectable ligand is a ligand conjugated to a detectable marker, the 
ligand is conjugated to the detectable marker before the cells are 
contacted with the ligand. If the detectable ligand is a ligand permitting 
detection through contact with a second detectable ligand, in one 
embodiment the ligand is contacted with the second detectable ligand 
before the cells are contacted with the ligand. In another embodiment, the 
ligand is contacted with the second detectable ligand after the cells are 
contacted with the ligand but before the cells are preserved. In still 
another embodiment, the ligand is contacted with the second detectable 
ligand after the cells in the sample are preserved. 
Conditions permitting the known ligand to specifically form a complex with 
the moiety recognized thereby are well known in the art. In one 
embodiment, the cells are contacted with the known ligand at a temperature 
of from about 0.degree. C. to about 10.degree. C. In another embodiment, 
the cells are contacted with the known ligand at a temperature of from 
about 0.degree. C. to about 4.degree. C. In another embodiment, the cells 
are contacted with the known ligand at a temperature of about 4.degree. C. 
In still another embodiment, the cells are contacted with the known ligand 
at a temperature which is physiologically compatible with the cells, for 
example a temperature of about 37.degree. C. 
Any method for detecting the presence of labelled cells known in the art 
may be used in the subject method for detecting the presence of rare cells 
in a sample which specifically possess on their surfaces a moiety 
recognized by a known ligand. The method for detecting the presence of 
labelled cells may be chosen based on the particular type of detectable 
ligand used. Examples of methods for detecting labelled cells which may be 
used in the subject method include, but are not limited to, immunoaffinity 
chromatography when the detectable ligand is an antibody, separation by 
magnetic beads when the detectable ligand is a magnetically charged 
substance, and fluorescence activated cell sorting when the detectable 
ligand is conjugated to a fluorophore. A further example of a method for 
detecting labelled cells which may be used in the subject method is 
visualization by light microscopy. 
The above-described method for detecting rare cells present in a sample may 
further comprise quantitatively determining any labelled cells detected in 
step (e). Any method for quantitatively determining cells may be used in 
the subject method. Methods for quantitatively determining cells are 
well-known to those of ordinary skill in the art and include, but are not 
limited to, counting cells with a fluorescence activated cell sorter as 
well as using immunoturbidity assays. 
The above-described method for detecting rare cells present in a sample may 
further comprise isolating any labelled cells detected in step (e). Any 
method for isolating labelled cells known in the art may be used in the 
subject method. The method for isolating labelled cells may be chosen 
based on the particular type of detectable ligand used. Examples of 
methods for isolating cells which may be used in the subject method 
include, but are not limited to, immunoaffinity chromatography when the 
detectable ligand is an antibody, separation by magnetic beads when the 
detectable ligand is a magnetically charged substance, and fluorescence 
activated cell sorting when the detectable ligand is conjugated to a 
fluorophore. 
In another embodiment of the above-described method for detecting the 
presence of rare cells present in a sample, the method further comprises 
isolating any cells which are not detected in step (e). Isolation of cells 
which are not detected in step (e) may be accomplished by excluding the 
labelled cells detected in step (e). The method for excluding labelled 
cells may be chosen based on the particular type of detectable ligand 
used. Examples of methods for excluding labelled cells which may be used 
in the subject method include, but are not limited to, immunoaffinity 
chromatography when the detectable ligand is an antibody, separation by 
magnetic beads when the detectable ligand is a magnetically charged 
substance, and fluorescence activated cell sorting when the detectable 
ligand is conjugated to a fluorophore. 
This invention further provides a method for determining whether a fetus is 
afflicted with a genetic abnormality which comprises the steps of (a) 
obtaining a sample comprising fetal cells from the fetus, which sample 
comprises fetal cells which specifically possess on their surfaces a 
moiety recognized by a known ligand; (b) detecting the presence of fetal 
cells in the sample so obtained according to the method for detecting the 
presence of rare cells in a sample described above; and (c) determining 
whether the fetal cells so detected are characteristic of cells from a 
fetus having the genetic abnormality, so as to thereby determine whether 
the fetus is afflicted with the genetic abnormality. 
Any method for obtaining a sample comprising fetal cells from a fetus may 
be used in the subject invention, and such methods are well-known in the 
art. Methods for obtaining a sample comprising fetal cells from a fetus 
well-known in the art include, but are not limited to, withdrawing a 
sample of peripheral blood from the mother bearing the fetus. 
Any method for determining whether fetal cells are characteristic of cells 
from a fetus possessing a genetic abnormality may be used in the subject 
method. Such methods are well-known to those of ordinary skill in the art. 
For example, fetal cells characteristic of cells from a fetus having Down 
syndrome may be determined by contacting the preserved fetal cells with a 
fluorescently-labeled nucleic acid probe specific for chromosome 21 and 
visualizing the cells by fluorescence in situ hybridization. The presence 
of three chromosome 21-specific signals indicates the fetus has Down 
syndrome. As another example, fetal cells characteristic of cells from a 
fetus having Turner syndrome may be determined by contacting the preserved 
fetal cells with a fluorescently-labeled X-chromosome specific nucleic 
acid probe and a fluorescently-labeled Y-chromosome specific nucleic acid 
probe, followed by visualizing the cells by fluorescence in situ 
hybridization. The presence of a single X-chromosome (with no 
Y-chromosome) indicates the fetus has Turner syndrome. 
In one embodiment of the subject method for determining whether a fetus is 
afflicted with a genetic abnormality, the fetal cells detected in step (b) 
are isolated prior to determining whether they are characteristic of cells 
from a fetus having the genetic abnormality in step (c). Any method for 
isolating cells may be used and will be selected based on the particular 
detectable ligand used in the method for detecting fetal cells in step 
(b). Such methods for isolating cells are well-known to those of ordinary 
skill in the art as discussed above. 
This invention also provides a method for determining the gender of a fetus 
which comprises the steps of (a) obtaining a sample comprising fetal cells 
from the fetus, which sample comprises fetal cells which specifically 
possess on their surfaces a moiety recognized by a known ligand; (b) 
detecting the presence of fetal cells in the sample so obtained according 
to the method for detecting the presence of rare cells in a sample 
described above; and (c) determining whether the preserved, labelled, 
fetal cells in the sample so obtained possess two X chromosomes or an X 
and a Y chromosome, so as to thereby determine the gender of the fetus. 
Any method for determining whether fetal cells possess two X chromosomes or 
an X and a Y chromosome may be used in the subject method. Such methods 
are well-known to those of ordinary skill in the art and include, but are 
not limited to, contacting cells with a detectable nucleic acid probe 
specific for a DNA sequence present only on an X chromosome and a 
detectable nucleic acid probe specific for a DNA sequence only on a Y 
chromosome under conditions suitable to permit hybridization of the probes 
to the DNA sequences if present within the nuclei of the cells and 
subsequently detecting the presence of any hybridized probes. The presence 
of two X chromosomes indicates the fetus is a normal female. The presence 
of an X and a Y chromosome indicates that the fetus is a normal male. 
In one embodiment of the subject method for determining the gender of a 
fetus, the fetal cells detected in step (b) are isolated prior to 
determining whether they possess two X chromosomes or an X and a Y 
chromosome in step (c). Any method for isolating cells may be used and 
will be selected based on the particular detectable ligand used in the 
method for detecting fetal cells in step (b). Such methods for isolating 
cells are well-known to those of ordinary skill in the art as discussed 
above. 
Also provided is a method of determining whether a subject is afflicted 
with a tumor normally tending to shed cells into the blood of a subject 
afflicted therewith which comprises the steps of (a) obtaining a blood 
sample from the subject; (b) removing erythrocytes from the blood sample 
so obtained; and (c) detecting the presence of tumor cells in the 
resulting sample according to the method for detecting the presence of 
rare cells in a sample which specifically possess on their surfaces a 
moiety recognized by a known ligand described above, so as to thereby 
determine whether the subject is afflicted with said tumor. 
Tumors normally tending to shed cells into the blood of a subject afflicted 
therewith are well-known in the art as described above and include, but 
are not limited to, melanomas, breast tumors, spleen tumors, liver tumors, 
and kidney tumors. 
Any method for obtaining a blood sample from a subject may be used in the 
subject method, and such methods are well-known to those of ordinary skill 
in the art. 
Any method for removing erythrocytes from a blood sample may be used in the 
subject method, and such methods are well-known to those of ordinary skill 
in the art as described above. 
This invention further provides a method of detecting in a subject the 
presence of cells expressing a malignant phenotype which comprises the 
steps of (a) obtaining a suitable sample from the subject; (b) removing 
erythrocytes from the sample so obtained; and (c) detecting the presence 
of cells expressing the malignant phenotype in the resulting sample 
according to the method for detecting the presence of rare cells in a 
sample which specifically possess on their surfaces a moiety recognized by 
a known ligand described above, so as to thereby detect in the subject the 
presence of cells expressing the malignant phenotype. 
Cells expressing a malignant phenotype are well-known in the art as 
described above. As used herein, a "suitable sample" is a sample in which 
cells expressing a malignant phenotype would be expected in an afflicted 
subject. Such samples are well-known in the art and include, but are not 
limited to, biopsies, tissue aspirates, lavages, and biological fluid 
samples such as blood or urine. Any method for removing erythrocytes from 
a sample may be used in the subject method. 
In one embodiment, the suitable sample is obtained from a subject afflicted 
with a tumor. In another embodiment, the suitable sample is obtained from 
a subject previously afflicted with a tumor which tumor has been removed. 
In a further embodiment, the suitable sample is obtained from a subject 
who has been treated with an anti-tumor therapy such as radiotherapy or 
chemotherapy. 
When the suitable sample is obtained from a subject previously afflicted 
with a tumor which tumor has been removed and characterized as benign or 
quiescent, the subject method for detecting in a subject the presence of 
cells expressing a malignant phenotype is useful, for example, for 
monitoring the transition of tumor cells remaining in the subject from a 
benign or quiescent phenotype to a malignant or aggressive phenotype. 
When the suitable sample is obtained from a subject who has been treated 
with an anti-tumor therapy, cells expressing a malignant phenotype 
detected according to the subject method may be further analyzed to 
determine whether they possess a genetic alteration associated with the 
anti-tumor therapy. Anti-tumor therapies resulting in genetic alterations 
in cells expressing a malignant phenotype which may be determined 
according to the subject invention include any anti-tumor therapy known to 
those of ordinary skill in the art. Anti-tumor therapies known in the art 
include, but are not limited to, radiotherapy and chemotherapy. 
Genetic alterations associated with radiotherapy include mutations which 
are known to increase sensitivity to radiation in cells. Accordingly, the 
method of the subject invention is useful, for example, for assessing 
whether radiotherapy will be cytotoxic to cells having a malignant 
phenotype in an afflicted subject, and consequently whether the benefits 
from radiotherapy will outweigh harm to the subject. Examples of genetic 
alterations associated with radiotherapy which are known to indicate 
increased sensitivity to radiation include, for example, possession of the 
XRCC-1 gene and increased expression of p53 protein. Possession of the 
XRCC-1 gene may be determined in cells, for example, by polymerase chain 
reaction or fluorescent in situ hybridization. Increased expression of p53 
in cells may be determined, for example, by using a flow cytometric assay. 
Genetic alterations associated with chemotherapy include genetic 
alterations associated with drug resistance. Accordingly, when a sample 
has been obtained from a subject who has been treated with an anti-tumor 
therapy which included chemotherapy, the subject method for detecting in a 
subject the presence of cells expressing a malignant phenotype is useful, 
for example, for detecting drug resistance in the subject. "Drug 
resistance" occurs when cells possessing a malignant phenotype remain in a 
subject after a regimen of chemotherapy, said cells being resistant to the 
therapy. Drug resistance includes multidrug resistance, which occurs when 
cells possessing a malignant phenotype remaining in a subject after a 
regimen of chemotherapy express a multidrug resistant phenotype, i.e. a 
phenotype associated with increased expression of mdr genes. As used 
herein, "a regimen of chemotherapy" includes treatment with a single 
chemotherapeutic agent, as well as treatment with more than one 
chemotherapeutic agent. 
Accordingly, this invention also provides a method for determining whether 
a subject who has been treated with an anti-tumor therapy, said therapy 
having included chemotherapy, possesses cells expressing a malignant 
phenotype which have undergone a genetic alteration associated with drug 
resistance, which comprises the steps of (a) detecting in the subject the 
presence of cells expressing a malignant phenotype according to the method 
described above and; (b) determining whether any cells so detected are 
characteristic of cells which have undergone a genetic alteration 
associated with drug resistance, thereby determining whether the subject 
possesses cells expressing a malignant phenotype which have undergone a 
genetic alteration associated with drug resistance. 
Genetic alterations associated with drug resistance are well-known in the 
art. Examples of genetic alterations associated with drug resistance 
include up-regulation or amplification of mdr genes, which are 
characteristic of multidrug resistance, or amplification of the gene 
encoding for dihydrofolate reductase. 
Means for determining whether cells are characteristic of cells which have 
undergone a particular genetic alteration which is associated with drug 
resistance are known to those of ordinary skill in the art. For example, 
if the genetic alteration is amplification of mdr genes, cells having this 
genetic alteration may be identified by in situ hybridization with nucleic 
acid probes complementary to mdr genes. Amplification of mdr genes will be 
indicated by the quantity of probes which hybridize to the cells. If the 
genetic alteration is the amplification of the gene encoding for 
dihydrofolate reductase, cells characteristic of such a genetic alteration 
may be identified by in situ hybridization with a nucleic acid probe 
complementary to the gene encoding for dihydrofolate reductase. 
Amplification of the gene encoding for dihydrofolate reductase may be 
determined by the quantity of probe which hybridizes to the cells. 
In one embodiment of the subject method for determining whether a subject 
who has been treated with an anti-tumor therapy having included 
chemotherapy possesses cells expressing a malignant phenotype which have 
undergone a genetic alteration associated with drug resistance, the cells 
expressing the malignant phenotype detected in step (a) are isolated prior 
to determining whether they are characteristic of cells which have 
undergone a genetic alteration associated with drug resistance in step 
(c). Any method for isolating cells may be used and will be selected based 
on the particular detectable ligand used in the method for detecting the 
cells expressing a malignant phenotype in step (a). Such methods for 
isolating cells are well-known to those of ordinary skill in the art as 
discussed above. 
Once cells characteristic of a particular genetic alteration associated 
with drug resistance are identified, the treatment of the subject may be 
modified so as to destroy any remaining drug resistant cells, for example 
by treating the subject with a chemical against which the remaining drug 
resistant cells are sensitive.