Systems and methods for identifying rare cells
Disclosed herein are compositions and methods of fixing and staining rare cells. Further, disclosed herein are methods of identifying circulating tumor cells (CTC). In some embodiments, the method includes: imaging a cell sample to identify a cell of interest; determining a first pixel intensity of a stained nuclear area; determining a second pixel intensity of a background area; calculating a ploidy status of the cell of interest by subtracting the second pixel intensity from the first pixel intensity; and determining whether the cell of interest is a CTC based on the ploidy status. The method may be computer implemented, such that the method uses a machine learning algorithm to identify a feature; process the feature to extract a parameter of interest; analyze the parameter of interest; and when the parameter of interest is greater than or less than a predetermined threshold, classify the cell of interest as a CTC.
1. A reagent system for fixing cells, the reagent system comprising:
a first fixing buffer comprising:
at least 3% w/v of polyvinylpyrrolidone diluted in methanol;
a second fixing buffer comprising:
at least 5% v/v of glycerol,
at least 0.01% v/v of a detergent, wherein the detergent comprises a polysorbate surfactant, polysorbate 20, saponin, Triton X-100, Triton X-114, Tween-40, Tween-80, digitonin, CHAPS, CHAPSO, n-dodecyl-β-D-maltoside, sodium dodecyl sulfate, or a combination thereof, and
at least 0.005% w/v of a chrome alum, wherein the glycerol, detergent, and chrome alum are diluted in saline; and
a blocking buffer comprising:
at least 1% v/v of one of polyvinylpyrrolidone or glycerol;
at least 0.01% v/v of a detergent, wherein the detergent comprises a polysorbate surfactant, polysorbate 20, saponin, Triton X-100, Triton X-114, Tween-40, Tween-80, digitonin, CHAPS, CHAPSO, n-dodecyl-β-D-maltoside, sodium dodecyl sulfate, or a combination thereof; and
at least 0.1% w/v hydrolyzed collagen, wherein the one of polyvinylpyrrolidone or glycerol, detergent, and hydrolyzed collagen are diluted in saline.
2. A reagent system for fixing cells, the reagent system comprising:
a first fixing buffer comprising:
3% to 20% w/v of polyvinylpyrrolidone diluted in methanol; and
a second fixing buffer comprising:
5% to 30% v/v of glycerol,
0.01% to 1% v/v of a detergent, wherein the detergent comprises a polysorbate surfactant, polysorbate 20, saponin, Triton X-100, Triton X-114, Tween-40, Tween-80, digitonin, CHAPS, CHAPSO, n-dodecyl-β-D-maltoside, sodium dodecyl sulfate, or a combination thereof, and
0.005% to 1% w/v of a chrome alum, wherein the glycerol, detergent, and chrome alum are diluted in saline; and
a blocking buffer comprising:
at least 1% v/v of one of polyvinylpyrrolidone or glycerol;
at least 0.01% v/v of a detergent, wherein the detergent comprises a polysorbate surfactant, polysorbate 20, saponin, Triton X-100, Triton X-114, Tween-40, Tween-80, digitonin, CHAPS, CHAPSO, n-dodecyl-β-D-maltoside, sodium dodecyl sulfate, or a combination thereof; and
at least 0.1% w/v hydrolyzed collagen, wherein the one of polyvinylpyrrolidone or glycerol, detergent, and hydrolyzed collagen are diluted in saline.
3. The reagent system of claim 1 , the reagent system comprising:
a first fixing buffer comprising:
5% w/v of polyvinylpyrrolidone diluted in methanol; and
a second fixing buffer comprising:
15% v/v of glycerol,
0.4% v/v of a detergent, wherein the detergent comprises a polysorbate surfactant, polysorbate 20, saponin, Triton X-100, Triton X-114, Tween-40, Tween-80, digitonin, CHAPS, CHAPSO, n-dodecyl-β-D-maltoside, sodium dodecyl sulfate, or a combination thereof, and
0.01% w/v of a chrome alum, wherein the glycerol, detergent, and chrome alum are diluted in saline; and
a blocking buffer comprising:
at least 1% v/v of one of polyvinylpyrrolidone or glycerol;
at least 0.01% v/v of a detergent, wherein the detergent comprises a polysorbate surfactant, polysorbate 20, saponin, Triton X-100, Triton X-114, Tween-40, Tween-80, digitonin, CHAPS, CHAPSO, n-dodecyl-β-D-maltoside, sodium dodecyl sulfate, or a combination thereof; and
at least 0.1% w/v hydrolyzed collagen, wherein the one of polyvinylpyrrolidone or glycerol, detergent, and hydrolyzed collagen are diluted in saline.
4. The reagent system of claim 1 , further comprising at least 0.01M glycine.
5. The reagent system of claim 1 , wherein the blocking buffer comprises:
1% to 50% v/v of one of polyvinylpyrrolidone or glycerol;
0.01% to 2% v/v of a detergent, wherein the detergent comprises a polysorbate surfactant, polysorbate 20, saponin, Triton X-100, Triton X-114, Tween-40, Tween-80, digitonin, CHAPS, CHAPSO, n-dodecyl-β-D-maltoside, sodium dodecyl sulfate, or a combination thereof; and
0.1% to 10% w/v hydrolyzed collagen, wherein the one of polyvinylpyrrolidone or glycerol, detergent, and hydrolyzed collagen are diluted in saline.
6. The reagent system of claim 5 , further comprising 0.01M to 1M glycine.
7. The reagent system of claim 1 , wherein the blocking buffer comprises:
15% v/v of one of polyvinylpyrrolidone or glycerol;
0.4% v/v of a detergent, wherein the detergent comprises a polysorbate surfactant, polysorbate 20, saponin, Triton X-100, Triton X-114, Tween-40, Tween-80, digitonin, CHAPS, CHAPSO, n-dodecyl-β-D-maltoside, sodium dodecyl sulfate, or a combination thereof; and
2% w/v hydrolyzed collagen, wherein the one of polyvinylpyrrolidone or glycerol, detergent, and hydrolyzed collagen are diluted in saline.
8. The reagent system of claim 7 , further comprising 0.3M glycine.
9. The reagent system of claim 1 , wherein the hydrolyzed collagen is pig derived.
10. A method for fixing a cell, the method comprising:
applying a first fixing buffer to the cell at a temperature colder than −5° C., the first fixing buffer comprising:
3% to 20% w/v of polyvinylpyrrolidone diluted in methanol;
applying a second fixing buffer to the cell, the second fixing buffer comprising:
5% to 30% v/v of glycerol,
0.01% to 1% v/v of a detergent, wherein the detergent comprises a polysorbate surfactant, polysorbate 20, saponin, Triton X-100, Triton X-114, Tween-40, Tween-80, digitonin, CHAPS, CHAPSO, n-dodecyl-β-D-maltoside, sodium dodecyl sulfate, or a combination thereof, and
0.005% to 1% w/v of a chrome alum, wherein the glycerol, detergent, and chrome alum are diluted in saline and
applying a blocking buffer to the cell, the blocking buffer comprising:
1% to 50% v/v of one of polyvinylpyrrolidone or glycerol,
0.01% to 2% v/v of a detergent, wherein the detergent comprises a polysorbate surfactant, polysorbate 20, saponin, Triton X-100, Triton X-114, Tween-40, Tween-80, digitonin, CHAPS, CHAPSO, n-dodecyl-β-D-maltoside, sodium dodecyl sulfate, or a combination thereof, and
0.1% to 10% w/v hydrolyzed collagen, wherein the one of polyvinylpyrrolidone or glycerol, detergent, and hydrolyzed collagen are diluted in saline.
11. The method of claim 10 , further comprising:
cytocentrifuging the cell onto a slide, wherein the cell is coated in a buffer comprising:
3% to 30% v/v of polyvinylpyrrolidone, and
0.005% to 1% w/v of chrome alum, wherein the polyvinylpyrrolidone and chrome alum are diluted in saline.
12. The method of claim 11 , wherein the slide is coated with gelatin.
13. The method of claim 12 , wherein the slide is further coated with chrome alum.
14. The method of claim 10 , wherein the cell is a circulating tumor cell.
15. The method of claim 10 , wherein the cell is embedded in a tissue section.
16. The method of claim 10 , further comprising:
staining the cell with a fluorophore-tagged antibody.