Methods for identification of particles in a fluid sample
A method of distinguishing between proteinaceous and non-proteinaceous particulates in a fluid sample includes the steps of acquiring a brightfield background image of a membrane filter, introducing a fluid sample onto the membrane filter, acquiring a brightfield image of filtered particles resting on the membrane filter, generating a particle mask based on the brightfield background image and the brightfield image of filtered particles, introducing a fluorescent dye onto the membrane filter, detecting fluorescence on the particle mask, and distinguishing between proteinaceous and non-proteinaceous particulates based on the detected fluorescence. A method for detecting other types of particles, such as polysorbate particles, silicone oil or protein monomers is also disclosed.
1. A method of distinguishing between particulates in a fluid sample comprising:
acquiring a brightfield background image of a membrane filter while the membrane filter is free of a fluid sample;
introducing a fluid sample onto the membrane filter;
acquiring a brightfield image of filtered particles resting on the membrane filter;
introducing a fluorescent dye onto the membrane filter; and
distinguishing between particulates based on the brightfield background image, the brightfield image of filtered particles, and detected fluorescence.
2. The method of claim 1 further comprising:
generating a total particle distribution, a target particle distribution, and a non-target particle distribution based on the distinguishing.
3. The method of claim 1 further comprising:
generating an image of target and non-target particles, wherein the target and non-target particles are different colors.
4. The method of claim 1 further comprising:
acquiring a background fluorescence and removing baseline fluorescent intensity.
5. The method of claim 1 , wherein the step of introducing a fluorescent dye takes place immediately after the step of introducing a fluid sample onto the membrane filter.
6. The method of claim 1 , wherein the fluorescent dye is introduced to the fluid sample prior to the step of introducing the fluid sample onto the membrane filter.
7. The method of claim 1 , wherein the membrane filter is a track etched membrane.
8. The method of claim 1 , wherein the membrane filter is dyed with a fluorescence photo absorber.
9. The method of claim 8 , wherein the photo absorber comprises sudan black dye.
10. The method of claim 1 , wherein the membrane filter comprises a polycarbonate or polyester.
11. The method of claim 1 , wherein the membrane filter is coated with polyvinylpyrrolidone.
12. The method of claim 1 , wherein the step of introducing a fluorescent dye comprises introducing between 5 and 50 uL of dye.
13. The method of claim 1 , wherein the fluid sample is bounded by a hydrophobic ring disposed on a well plate.