Detection of fetal cells from maternal blood

Abstract

The present application relates to methods for identification of foetal cells and generation and isolation of binding members recognising foetal cells. Said methods may further be used for other purposes relating to characterisation of biological samples and biological antigens. The methods are characterised by the applicability in situations where the interesting objects are present in a limited amount, or where the interesting objects are intermixed with other material, thus the methods are suitable for use in situations where the ratio of the interesting material compared to other material is low. The application discloses methods for use of detecting foetal cells and method of generating/isolating binding members towards antigenic material of low abundancy.

1 . A method of detecting a fetal cell in a maternal blood sample comprising the following steps of:
 a. providing a maternal blood sample,   b. fixing and permealizing nucleated cells present in said blood sample while maintaining cell morphology, maintaining protein content of the cells and ensuring accessibility for a hybridization probe.   c. adding a hybridization probe, and   d. detecting at least one fetal cell.   
   
   
       2 . The method according to  claim 1 , comprising a pre-fixation step. 
   
   
       3 . The method according to  claim 2 , wherein the pre-fixation step comprises PFA treatment. 
   
   
       4 . The method according to  claim 3 , wherein the pre-fixation step comprises incubation of cells in a solution comprising 0.1-4% PFA. 
   
   
       5 . The method according to  claim 1 , wherein the fixation and permeabilization step comprises methanol and acetone treatment. 
   
   
       6 . The method according to  claim 1 , comprising a re-fixation step. 
   
   
       7 . The method according to  claim 6 , wherein the re-fixation step comprises PFA treatment. 
   
   
       8 . The method according to  claim 7 , wherein the re-fixation step comprises incubation of cells in a solution comprising 1-5% PFA. 
   
   
       9 . The method according to  claim 1 , comprising a dehydration steps in ethanol. 
   
   
       10 . The method according to  claim 1 , comprising an enrichment step. 
   
   
       11 . The method according to  claim 10 , wherein the enrichment step does not discriminate between different nucleated cell types. 
   
   
       12 . The method according to  claim 11 , wherein the enrichment step comprises a step of erythrocyte lysis. 
   
   
       13 . The method according to  claim 12 , wherein erythrocyte lysis is obtained by NH 4 Cl mediated lysis. 
   
   
       14 . The method according to  claim 1 , wherein the fetal cell is detected by selective labelling of DNA in the fetal cell. 
   
   
       15 . The method according to  claim 14 , wherein cell type specific DNA is selectively labelled by a hybridization technique. 
   
   
       16 . The method according to  claim 1 , wherein the fetal cell is detected by the presence of cell type specific epigenetic characteristics. 
   
   
       17 . The method according to  claim 1 , wherein the fetal cell is detected by selective labelling of cell type specific RNA in the fetal cell. 
   
   
       18 . The method according to  claim 17 , wherein cell type specific RNA is selective labelled by a hybridization technique. 
   
   
       19 . The method according to  claim 18 , wherein the cell type specific RNA is an mRNA. 
   
   
       20 . The method according to  claim 1 , wherein the fetal cell is detected by selective labelling of at least one cell type specific protein. 
   
   
       21 . The method according to  claim 1 , wherein the fetal cell is detected by morphological characteristics. 
   
   
       22 . The method according to  claim 1 , wherein the detected fetal cell is a male fetal cell. 
   
   
       23 . The method according to  claim 22 , wherein the male fetal cell is detected by reverse colour color FISH. 
   
   
       24 . The method according to  claim 1 , wherein the at least one fetal cell is detected using an automated scanner system. 
   
   
       25 . A fetal cell identified by the method according to  claim 1 . 
   
   
       26 . A binding member specifically recognizing the fetal cell according to  claim 25 . 
   
   
       27 . A fetal cell antigen recognized by the binding member according to  claim 26 . 
   
   
       28 . A method of isolating binding members recognizing target antigenic material of limited availability comprising the steps:
 a. identifying the target antigenic material,   b. contacting said target antigenic material with a binding member generating system and   c. isolating binding member(s) recognizing the target antigenic material.   
   
   
       29 .- 46 . (canceled) 
   
   
       47 . A fetal cell antigen recognized by a binding member isolated using the method of  claim 28 . 
   
   
       48 . (canceled) 
   
   
       49 . An assay method comprising the steps of:
 a. providing a maternal blood sample,   b. selectively labelling at least one fetal cell by labelling of a fetal cell antigen according to  claim 47     in said blood sample.   
   
   
       50 . An assay method comprising the steps of:
 a. providing a maternal blood sample,   b. selectively labelling at least one fetal cell by use of a binding member isolated using the method of  claim 28     in said blood sample.   
   
   
       51 .- 64 . (canceled)