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Non-invasive method for isolation and detection of fetal DNA

a fetal dna and non-invasive technology, applied in the field of non-invasive methods for isolation and detection of fetal dna, can solve the problems of not being able to distinguish fetal cells from maternal cells, not being able to confirm the presence of fetal dna in cells, and not being able to successfully isolate cells subsequently shown to contain fetal dna

Inactive Publication Date: 2007-01-18
CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006] The present invention is based, at least in part, on the discovery that fetal nucleated cells are present in the peripheral blood of a pregnant woman at a level which allows them to be useful in prenatal diagnostic methods. The method of the present invention is non-invasive because a peripheral blood sample from pregnant women, not fetal blood, is used as the source of the fetal DNA. The fetal DNA is derived from fetal nucleated cells present in the peripheral blood of a pregnant woman. The method of the present invention can be used to assess fetal characteristics (e.g. fetal sex and chromosomal abnormalities) or can be used to diagnose whether a fetus has a prenatal disease at an early stage of the gestational period. The non-invasive method of the present invention does not expose the fetus or mother to risks, e.g. infection, fetal injury, and miscarriage, associated with invasive methods such as amniocentesis.

Problems solved by technology

There have been numerous reports of efforts to separate fetal cells from maternal cells present in maternal blood, but none has been successful in isolating cells subsequently shown to contain fetal DNA.
Distinguishing fetal cells from maternal cells has not been successful for several reasons, including the small number of fetal cells in a maternal blood sample and the fact that morphological differences are slight.
However, although cells sorted in this manner have been said to be of fetal origin, based on analysis of cell surface antigens, morphology, or cytogenetic criteria, there has not been confirmation that the cells contain fetal DNA.

Method used

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  • Non-invasive method for isolation and detection of fetal DNA
  • Non-invasive method for isolation and detection of fetal DNA
  • Non-invasive method for isolation and detection of fetal DNA

Examples

Experimental program
Comparison scheme
Effect test

example 1

Antibody Selection for Isolation and Sorting of Fetal Nucleated Erythrocytes (NRBCs)

Removal of Maternal Leucocytes from Maternal Blood Using Human Leucocyte Antigen (HLe-1)

[0074] The technique of fetal NRBC isolation began with an initial Ficoll-Hypaque density gradient centrifugation to remove the tremendously high number of non-nucleated erythrocytes in maternal blood. Peripheral blood was centrifuged and separated into a supernatant layer containing platelets, a mononuclear cell layer, and an agglutinated pellet consisting of non-nucleated erythrocytes and granulocytes. The mononuclear cell layer consisted of lymphocytes, monocytes, possible trophoblasts, and, due to their increased size and density, NRBCs and some reticulocytes. While the Ficoll-Hypaque centrifugation represented an initial enrichment in the proportion of fetal NRBCs present in the maternal sample, flow cytometry and cell sorting was used to improve the purity of the isolated cell population.

[0075] The monon...

example 2

DNA Hybridization Studies in HLe-1 Negative Cells Sorted from Maternal Blood

[0082] To confirm fetal origin of the cells sorted as described in Example 1, Y chromosomal probes were used because it is the Y chromosome that is unquestionably fetal in origin. The assessments were designed to study whether the presence of Y chromosomal DNA in maternal blood as detected on autoradiographs performed antenatally correlated with the subsequent birth of a male infant.

DNA Isolation

[0083] HLe-1 negative cells from cord blood and pregnant women were sorted into test tubes. Conventional methods of DNA isolation at well as modification of cruder methods (Lau, Y-F, et al. Lancet, 1:14-16 (1984); McCabe, E. R. B., et al., Hum Genet., 75:213-216 (1987) were attempted without success in detecting Y chromosome derived bands on Southern Blots. All were limited by the small numbers of cells present.

example 3

Direct Hybridization to Cells Deposited on Filters

[0084] In order to circumvent technical problems associated wtih DNA isolation, a method of direct DNA hybridization to cells flow sorted onto nitrocellulose filters was developed (Bianchi, D. W., et al., Cytometry, 8:197-202 (1987)). In control experiments, the sex of a newborn was determined from as few as 50 sorted cord blood leucocytes or 5,000 HLe-1 negative cells (a mixture of nucleated and non-nucleated cells).

[0085] The methodology was then applied to detection of Y chromosomal sequences in HLe-1 negative cells sorted from peripheral blood samples in 40 women between 8½ and 38 weeks gestation. Results were the following:

DeliveredDeliveredLost ToDot Blot HybridizationMaleFemaleFollow-with Y Chromosomal ProbeInfantInfantup+320−21122

[0086] It was concluded that hybridization with this probe was not predictive of male pregnancy. The possibility exists that there was fetal DNA present on the filters where DNA hybridization occ...

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Abstract

A method of detecting the presence or absence of the fetal DNA sequence of interest in fetal DNA derived from a sample of peripheral blood obtained from a pregnant woman is described. The method involves obtaining a sample peripheral blood from a pregnant woman, treating the sample of peripheral blood such that the fetal DNA present in the fetal nucleated cells is made available for detection and detecting the presence or absence of the fetal DNA sequence of interest in the available fetal DNA. The proportion of fetal nucleated cells present in the sample of peripheral blood can be increased forming a sample enriched in fetal nucleated cells prior to the detection step. The fetal DNA sequence of interest can be detected by treating the peripheral blood sample such that fetal DNA present in the sample is made available for hybridization with a DNA probe and subsequently contacting the available fetal DNA with a DNA probe hybridizable to fetal DNA of interest under hybridization conditions. The presence or absence of hybridization between the DNA probe and the fetal DNA of interest is detected as an indication of the presence or absence of the fetal DNA of interest.

Description

RELATED APPLICATIONS [0001] This application is a continuation-in-part application of U.S. Ser. No. 07 / 772,689, filed on Oct. 7, 1991, which is a continuation-in-part application of U.S. Ser. No. 07 / 706,393, filed on May 28, 1991, now abandoned, which is a continuation-in-part of U.S. Ser. No. 07 / 436,057, filed on Nov. 13, 1989, now abandoned, all being entitled “Non-Invasive Method for Isolation and Detection of Fetal DNA” by Diana W. Bianchi. The contents of all of the forementioned applications are hereby expressly incorporated by reference.FUNDING [0002] Work described herein was supported by the National Institute of Health and Children's Hospital Medical Center. BACKGROUND [0003] A variety of fetal cell types—platelets, trophoblasts, erythrocytes and leucocytes—cross the placenta and circulate transiently within maternal blood (Schroder, J., J. Med. Genet. 12:230-242 (1975); Douglas G. W. et al., Am. J. Obstet. Gynec., 78:960-973 (1959)). There have been numerous reports of ef...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34G01N33/50G01N33/569G01N33/80
CPCC12Q1/6804C12Q2600/156C12Q1/6841C12Q1/686C12Q1/6876C12Q1/6879C12Q1/6881C12Q1/6883G01N33/5002G01N33/56966G01N33/56972G01N33/56977G01N33/80C12Q1/6806G01N2015/1006C12Q2547/00C12Q2545/113C12Q2543/10C12Q2565/626C12Q2565/137
Inventor BIANCHI, DIANA W.
Owner CHILDRENS HOSPITAL
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