Claims for Patent: 9,447,467

Summary for Patent: 9,447,467

Title:	Methods for obtaining fetal genetic material
Abstract:	The present invention relates to a method of enriching fetal nuclei from a sample. Enriched fetal nuclei can be used in a variety of procedures including, detection of a trait of interest such as a disease trait, or a genetic predisposition thereto, gender typing and parentage testing.
Inventor(s):	Allman; Richard (Wyndham Vale, AU), Mantzaris; Debbie (Avondale Heights, AU), Vom; Eduardo (Richmond, AU), Lewis; Craig Matthew (Eltham, AU)
Assignee:	GENETIC TECHNOLOGIES LIMITED (AU)
Application Number:	13/265,485
Patent Claims:	1. A method of enriching free fetal nuclei from a cervical mucous sample, comprising size-selecting cellular material which is less than about 10 .mu.m in size from a cervical mucous sample from a pregnant female, wherein the cervical mucous sample comprises intact maternal cells, intact fetal cells, and free fetal nuclei and wherein the cervical mucous sample is partially disaggregated prior to the size-selecting, thereby enriching the free fetal nuclei in the selected cellular material which is less than about 10 .mu.m in size. 2. The method of claim 1 which comprises selecting cellular material which is less than about 8 .mu.m in size. 3. The method of claim 1, wherein the cellular material is selected using at least one method selected from the group consisting of a cell strainer, flow cytometry, and microfluidics. 4. The method of claim 1, wherein the cervical mucous sample is partially mechanically and/or enzymatically disaggregated prior to the size-selecting. 5. The method of claim 4, wherein partially mechanically disaggregating the sample comprises gentle pipetting using an about 1 ml pipette and/or tweezing the sample apart using forceps. 6. The method of claim 4, wherein partially enzymatically disaggregating the sample comprises contacting the sample with a collagenase, a protease or a combination thereof. 7. The method of claim 1, further comprising selecting fetal cells. 8. The method of claim 7, wherein the method further comprises: i) at least partially mechanically disaggregating the sample to produce a cellular material suspension; ii) filtering the suspension through a first cell strainer which has a mesh size of at least about 100 .mu.m and collecting the cellular material that passed through the first cell strainer; iii) filtering the cellular material collected in step ii) through a second cell strainer which has a mesh size of less than about 40 .mu.m and collecting the cellular material that did not pass through the second cell strainer, and independently collecting the cellular material that passed through the second cell strainer; iv) filtering the cellular material that passed through the second cell strainer collected in step iii), through a third cell strainer which has a mesh size of less than about 10 .mu.m and collecting the cellular material that passed through the third cell strainer; and v) combining the cellular material that did not pass through the second cell strainer in step iii), which comprises fetal cells of a size greater than the mesh size of less than about 40 .mu.m, with the cellular material that passed through the third cell strainer in step iv), which comprises fetal nuclei of a size less than the mesh size of less than about 10 .mu.m. 9. The method of claim 7, wherein the method further comprises: i) at least partially enzymatically disaggregating the sample to produce a cellular material suspension; ii) filtering the suspension through a first cell strainer which has a mesh size of less than about 40 .mu.m and collecting the cellular material that did not pass through the first cell strainer, and independently collecting the cellular material that passed through the first cell strainer; iii) filtering the cellular material that passed through the first cell strainer in step ii), through a second cell strainer which has a mesh size of less than about 10 .mu.m and collecting the cellular material that passed through the second cell strainer; and iv) combining the cellular material that did not pass through the first cell strainer in step ii), which comprises fetal cells of a size greater than the mesh size of less than about 40 .mu.m, with the cellular material that passed through the second cell strainer in step iii), which comprises fetal nuclei of a size less than the mesh size of less than about 10 .mu.m. 10. The method of claim 7, wherein the method further comprises: i) at least partially mechanically disaggregating the sample to produce a cellular material suspension; ii) filtering the suspension through a cell strainer which has a mesh size of at least about 100 .mu.m and collecting the cellular material that passed through the cell strainer; iii) sorting the cellular material that passed through the cell strainer in step ii) by fluorescent activated cell separation (FACS) based on forward scatter and collecting cellular material which is at least about 40 .mu.m in size; iv) sorting the cellular material that passed through the cell strainer in step ii) and/or the cellular material which is at least about 40 .mu.m in size, collected in step iii), by fluorescent activated cell separation (FACS) based on forward scatter and collecting cellular material which is less than about 10 .mu.m; and v) combining the cellular material that is at least about 40 .mu.m in size, collected in step iii) and which comprises fetal cells, with the cellular material that is less than about 10 .mu.m, collected in step iv), and which comprises fetal nuclei. 11. The method of claim 7, wherein the method further comprises: i) at least partially mechanically and/or enzymatically disaggregating the sample to produce a cellular material suspension; ii) sorting the suspension by fluorescent activated cell separation (FACS) based on forward scatter and collecting cellular material which is between about 40 .mu.m and 100 .mu.m in size, and collecting cellular material which is less than about 10 .mu.m; and iii) combining the cellular material which is between about 40 .mu.m and 100 .mu.m in size, which comprises fetal cells, with the cellular material which is less than about 10 .mu.m and which comprises fetal nuclei. 12. The method of claim 7 which comprises positively selecting fetal cells using an agent which binds fetal cells but not maternal cells. 13. The method of claim 7, wherein the step of selecting comprises negatively selecting fetal cells using an agent which binds maternal cells but does not bind fetal cells. 14. The method of claim 13 where the method further comprises combining the fetal nuclei and fetal cells. 15. The method of claim 1, wherein the sample was obtained within 5 to 18 weeks of pregnancy. 16. The method of claim 1 wherein the sample is obtained from a pregnant female prior to the step of selecting. 17. The method of claim 1, wherein the cellular material is selected using a cell strainer or flow cytometry. 18. The method of claim 1, wherein the cellular material is selected using a cell strainer. 19. A method of enriching fetal cells and free fetal nuclei, comprising: i) at least partially mechanically and/or enzymatically disaggregating a cervical mucous sample from a pregnant female to produce a cellular material suspension comprising intact maternal cells, intact fetal cells and free fetal nuclei; ii) filtering the suspension through a first cell strainer which has a mesh size of at least about 100 .mu.m and collecting the cellular material that passed through the first cell strainer; iii) filtering the cellular material collected in step ii) through a second cell strainer which has a mesh size of less than about 40 .mu.m and collecting the cellular material that did not pass through the second cell strainer, and independently collecting the cellular material that passed through the second cell strainer; iv) filtering the cellular material that passed through the second cell strainer collected in step iii), through a third cell strainer which has a mesh size of less than about 10 .mu.m and collecting the cellular material that passed through the third cell strainer; and v) combining the cellular material that did not pass through the second cell strainer in step iii), which comprises fetal cells of a size greater than the mesh size of less than about 40 .mu.m, with the cellular material that passed through the third cell strainer in step iv), which comprises fetal nuclei of a size less than the mesh size of less than about 10 .mu.m; whereby the fetal cells and the free fetal nuclei are enriched.

Details for Patent 9,447,467

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2039-02-26