U.S. patent application number 10/113177 was filed with the patent office on 2002-09-26 for non-invasive method for isolation and detection of fetal dna.
This patent application is currently assigned to Children's Medical Center Corporation. Invention is credited to Bianchi, Diana W..
Application Number | 20020137088 10/113177 |
Document ID | / |
Family ID | 27503847 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020137088 |
Kind Code |
A1 |
Bianchi, Diana W. |
September 26, 2002 |
Non-invasive method for isolation and detection of fetal DNA
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.
Inventors: |
Bianchi, Diana W.;
(Brookline, MA) |
Correspondence
Address: |
Elizabeth A. Hanley, Esq.
Lahive & Cockfield, LLP
28 State Street
Boston
MA
02109
US
|
Assignee: |
Children's Medical Center
Corporation
|
Family ID: |
27503847 |
Appl. No.: |
10/113177 |
Filed: |
March 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10113177 |
Mar 28, 2002 |
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09827853 |
Apr 5, 2001 |
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09827853 |
Apr 5, 2001 |
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08470633 |
Jun 6, 1995 |
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08470633 |
Jun 6, 1995 |
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08338279 |
Nov 14, 1994 |
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5641628 |
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08338279 |
Nov 14, 1994 |
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07957736 |
Oct 7, 1992 |
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07957736 |
Oct 7, 1992 |
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07772689 |
Oct 7, 1991 |
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07772689 |
Oct 7, 1991 |
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07706393 |
May 28, 1991 |
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07706393 |
May 28, 1991 |
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07436057 |
Nov 13, 1989 |
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Current U.S.
Class: |
435/6.11 ;
435/91.2 |
Current CPC
Class: |
C12Q 2545/113 20130101;
C12Q 2547/00 20130101; C12Q 2565/137 20130101; C12Q 2565/137
20130101; C12Q 2545/113 20130101; C12Q 2547/00 20130101; C12Q
2543/10 20130101; C12Q 2543/10 20130101; C12Q 2565/626 20130101;
C12Q 1/686 20130101; C12Q 2565/626 20130101; G01N 2015/1006
20130101; G01N 33/56977 20130101; C12Q 1/6804 20130101; C12Q 1/6841
20130101; C12Q 1/6804 20130101; G01N 33/56966 20130101; C12Q 1/6881
20130101; C12Q 2600/156 20130101; C12Q 1/6876 20130101; C12Q 1/6841
20130101; G01N 33/56972 20130101; C12Q 1/6806 20130101; C12Q 1/6806
20130101; C12Q 1/6883 20130101; G01N 33/5002 20130101; C12Q 1/6879
20130101; C12Q 1/686 20130101; G01N 33/80 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
1. A method of detecting the presence or absence of a fetal DNA
sequence of interest in fetal DNA derived from a sample of
peripheral blood obtained from a pregnant woman, comprising:
obtaining a sample of peripheral blood from a pregnant woman;
treating the sample of peripheral blood such that fetal DNA present
in fetal nucleated cells present in the sample is made available
for detection resulting in available fetal DNA; and detecting the
presence or absence of a fetal DNA sequence of interest in the
available fetal DNA.
2. The method as claimed in claim 1 wherein the proportion of fetal
nucleated cells present in the sample of peripheral blood obtained
from the pregnant woman is increased forming a sample enriched in
fetal nucleated cells prior to the detection step.
3. The method of claim 1 wherein the sample enriched in fetal
nucleated cells is formed by separating the non-nucleated cells
from the nucleated in the peripheral blood sample forming a
nucleated cell enriched sample and by treating the nucleated cell
enriched sample such that the proportion of fetal cells in the
sample is increased forming a fetal nucleated cell enriched
sample.
4. The method of claim 1 wherein the fetal DNA is amplified prior
to the detection step resulting in amplified fetal DNA.
5. A method of detecting the presence or absence of fetal DNA of
interest in fetal DNA derived from a sample of peripheral blood
obtained from a pregnant woman, comprising: obtaining a sample of
peripheral blood from the woman; treating the sample of peripheral
blood such that fetal DNA present in fetal nucleated cells present
in the sample is made available for hybridization with a DNA probe
resulting in available fetal DNA; contacting the available fetal
DNA with a DNA probe hybridizable to fetal DNA of interest under
hybridization conditions; and detecting the presence or absence of
hybridization between the DNA probe and the fetal DNA of interest
as an indication of the presence or absence of the fetal DNA of
interest.
6. The method of claim 5 wherein the fetal nucleated cells in the
treating step are undifferentiated hematopoietic cells.
7. The method of claim 5 wherein the fetal nucleated cells are
selected from the group consisting of erythroblasts, lymphoblasts,
and myeloblasts.
8. The method of claim 6 wherein the undifferentiated hematopoietic
cells are erythroid cells.
9. The method of claim 8 wherein the undifferentiated erythroid
cells are fetal nucleated erythrocytes.
10. The method of claim 5 wherein the proportion of fetal nucleated
cells present in the sample of peripheral blood obtained from the
pregnant woman is increased forming a sample enriched in fetal
nucleated cells prior to the detection step.
11. The method of claim 10 wherein the sample enriched in fetal
nucleated cells is formed by separating non-nucleated cells from
nucleated cells in the peripheral blood sample forming a nucleated
cell enriched sample and by treating the nucleated cell enriched
sample such that the proportion of fetal cells in the sample is
increased forming a fetal nucleated cell enriched sample.
12. The method of claim 5 wherein the fetal DNA of interest in the
contacting and detecting steps is Y chromosomal DNA.
13. The method of claim 5 wherein the hybridization in the
detecting step is between the DNA probe and a disease causing
mutation.
14. The method of claim 13 wherein the disease causing mutation is
a cystic fibrosis-causing mutation.
15. The method of claim 13 wherein the disease causing mutation is
a Duchenne muscular dystrophy-causing mutation.
16. The method of claim 13 wherein the disease causing mutation is
a hemophilia A-causing mutation.
17. The method of claim 13 wherein the disease causing mutation is
a Gaucher disease-causing mutation.
18. The method of claim 13 wherein the disease causing mutation is
a sickle cell anemia-causing mutation.
19. The method of claim 13 wherein the hybridization in the
detecting step is between the DNA probe and a fetal DNA of interest
selected for assessing a chromosomal abnormality.
20. The method of claim 5 wherein the hybridization in the
detecting step is in situ hybridization.
21. The method of claim 6 wherein the hybridization in the
detecting step is in situ hybridization.
22. The method of claim 9 wherein the hybridization in the
detecting step is in situ hybridization.
23. The method of claim 12 wherein the hybridization in the
contacting step is in situ hybridization.
24. The method of claim 5 wherein the fetal DNA is amplified prior
to the detection step resulting in amplified fetal DNA.
25. The method of claim 6 wherein the fetal DNA is amplified prior
to the detection step resulting in amplified fetal DNA.
26. The method of claim 9 wherein the fetal DNA is amplified prior
to the detection step resulting in amplified fetal DNA.
27. The method of claim 3 further comprising determining the
gestational age at the time the sample of peripheral blood is
obtained and treating the sample such that fetal DNA present in a
selected type of fetal nucleated cells is made available for
hybridization, said type of fetal nucleated cell being selected
based upon the known presence of such fetal nucleated cells in the
peripheral blood of a pregnant woman at the determined gestational
age.
28. The method of claim 5 further comprising the step of treating
the peripheral blood sample to remove residual fetal nucleated
cells from a previous pregnancy prior to the detection step.
29. The method of claim 5 wherein the peripheral blood sample in
the obtaining step is obtained between about ten and about nineteen
weeks gestation.
30. A method of detecting a fetal DNA sequence of interest in fetal
DNA derived from a sample of peripheral blood obtained from a
pregnant woman, comprising: obtaining a sample of peripheral blood
from a pregnant woman; separating fetal nucleated cells from the
peripheral blood onto a solid support forming immobilized fetal
nucleated material; contacting the immobilized fetal nucleated
material with a DNA probe hybridizable to fetal DNA of interest
under hybridization conditions; and detecting the presence of
hybridization between the DNA probe and the fetal DNA of interest
as an indication of the presence or absence of the fetal DNA of
interest.
31. The method of claim 30 wherein the hybridization in the
detecting step is in situ hybridization.
32. The method of claim 31 wherein the hybridization of high copy
number repeat sequences present in the fetal DNA is suppressed in
the contacting step.
33. A method for determining the sex of a fetus, comprising:
obtaining a sample of peripheral blood from a woman pregnant with a
fetus; treating the sample of peripheral blood such that fetal DNA
present in fetal nucleated cells present in the sample is made
available for hybridization resulting in available fetal DNA;
contacting the available fetal DNA with a DNA probe hybridizable to
fetal Y chromosomal DNA under hybridization conditions; and
detecting the presence of hybridization between the DNA probe and
the fetal Y chromosomal DNA as an indication of a male fetua or the
absence of hybridization as an indication of a female fetus.
34. A method for diagnosing a disease in a fetus, comprising:
obtaining a sample of peripheral blood from a woman pregnant with a
fetus; treating the sample of peripheral blood such that fetal DNA
present in fetal nucleated cells present in the peripheral sample
is made available for hybridization resulting in available fetal
DNA; contacting the available DNA with a DNA probe hybridizable to
fetal DNA of interest associated with a disease under hybridization
conditions; and detecting the presence or absence of hybridization
between the DNA probe and the fetal DNA of interest associated with
the disease as an indication that the fetus has the disease.
35. A method for detecting a chromosomal abnormality in a fetus,
comprising: obtaining a sample of peripheral blood from a woman
pregnant with a fetus; separating fetal nucleated cells from the
peripheral blood sample onto a solid support forming immobilized
fetal nucleated material; contacting the immobilized fetal
nucleated materail with a DNA probe hybridizable to chromosomal
fetal DNA of interest under hybridization conditions; and detecting
the presence or absence of hybridization between the DNA probe and
the chromosomal fetal DNA of interest as an indication of the
presence or absence of a chromosomal abnormality.
36. The method of claim 35 wherein the hybridization in the
detecting step is in situ hybridization.
37. The method of claim 36 wherein the chromosomal abnormality is a
chromosomal aneuploidy.
38. The method of claim 37 wherein the chromosomal aneuploidy is
trisomy 21.
39. The method of claim 37 wherein the chromosomal aneuploidy is
trisomy 18.
40. The method of claim 37 wherein the chromosomal aneuploidy is
trisomy 13.
41. A method for determining whether a pregnancy is at risk,
comprising: obtaining a peripheral blood sample from a pregnant
woman at a selected gestational age; detecting the number of fetal
cells present in the peripheral blood sample; comparing the number
of fetal cells detected to a standard to determine whether the
number of fetal cells is indicative of a pregnancy at risk, said
standard being selected based on the number of fetal cells present
in a peripheral blood sample obtained from a woman having a normal
pregnancy at the selected gestational age.
42. A method of increasing the proportion of fetal nucleated cells
present in a peripheral blood sample containing nucleated and
non-nucleated cells, comprising: separating non-nucleated cells
from nucleated cells present in a peripheral blood sample forming a
nucleated cell enriched sample; and treating the nucleated cell
enriched sample such that the proportion of fetal cells in the
sample is increased forming a fetal nucleated cell enriched
sample.
43. The method of claim 42 wherein the separating step is a density
gradient centrifugation.
44. The method of claim 42 wherein the nucleated enriched sample is
treated by contacting the sample with (i) a first monoclonal
antibody which recognizes fetal nucleated cells but not maternal
cells and/or (ii) a second monoclonal antibody which recognizes
maternal cells but not fetal nucleated cells, under conditions
appropriate for antibody binding thereby producing (i) fetal
nucleated cell-first monoclonal antibody complexes and/or (ii)
maternal cell-second monoclonal antibody complexes, respectively,
and (i) separating fetal nucleated cell-first monoclonal antibody
complexes from maternal cells and/or (ii) separating maternal
cell-second monoclonal antibody complexes from fetal nucleated
cells thereby separating fetal nucleated cells from maternal
cells.
45. The method of claim 43 wherein the nucleated enriched sample is
treated by contacting the sample with (i) a first monoclonal
antibody which recognizes fetal nucleated cells but not maternal
cells and/or (ii) a second monoclonal antibody which recognizes
maternal cells but not fetal nucleated cells, under conditions
appropriate for antibody binding producing (i) fetal nulceated
cell-first monoclonal antibody complexes and/or (ii) maternal
cell-second monoclonal antibody complexes, respectively and, (i)
separating fetal nucleated cell-first monoclonal antibody complexes
from maternal cells and/or (ii) separating maternal cell-second
monoclonal antibody complexes from fetal nucleated cells thereby
separating fetal nucleated cells from maternal cells.
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 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 (e.g., trophoblasts are
the only fetal cells which can be distinguished from maternal cells
by morphology alone).
[0004] Others report screening the peripheral blood of pregnant
women for cells of fetal origin. Fetal identification relied on the
presence of a single cytogenetic marker, the Y chromosome.
Lymphocytes with a putative "XY" karyotype were found in the
maternal circulation as early as 14 weeks gestation (Walknowska,
J., et al., The Lancet, 1119-1122 (1979)).
[0005] The availability of flow cytometry has led many to suggest
that fetal cells could be obtained through the use of a flow
cytometer and that such cells could be exploited for prenatal
genetic diagnosis. 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. A method by
which fetal DNA could be obtained from maternal blood during
pregnancy would be valuable, particularly if it made it possible to
carry out prenatal diagnosis by a noninvasive technique.
DISCLOSURE OF THE INVENTION
[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.
[0007] The present invention pertains to a method of detecting the
presence or absence of a fetal DNA sequence of interest in fetal
DNA derived from a sample of peripheral blood obtained from a
pregnant woman. 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.
[0008] The method of the present invention can be used to determine
the sex of a fetus by contacting the peripheral blood sample from a
woman pregnant with a fetus with a DNA probe hybridizable to fetal
Y chromosomal DNA. The presence of hybridization between the DNA
probe and the fetal Y chromosomal DNA can be detected as an
indication of a male fetus or the absence of hybridization can be
detected as an indication of a female fetus.
[0009] The method of the present invention also may be used for
diagnosing a disease in a fetus. A sample of peripheral blood
obtained from a woman pregnant with a fetus is contacted with DNA
probe hybridizable to fetal DNA of interest associated with a
disease 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 whether the fetus has the
disease.
[0010] The method of the present invention further can be used to
detect a chromosomal abnormality in a fetus such as a chromosomal
aneuploidy, e.g., trisomy 13, trisomy 18, or trisomy 21. A sample
of peripheral blood from the woman pregnant with a fetus is
obtained. The fetal nucleated cells are separated from the
peripheral blood sample onto a solid support forming immobilized
fetal nucleated material, e.g. metaphase or interphase nuclei. The
immobilized fetal nucleated material is contacted with a DNA probe
hybridizable to chromosomal fetal DNA of interest under
hybridization conditions. The presence or absence of hybridization
between the DNA probe and the chromosomal fetal DNA of interest is
detected as an indication of the presence or absence of a
chromosomal abnormality.
[0011] The method of the present invention further can be used to
determine whether a pregnancy is at risk. Fetal blood hemhorrages
into the maternal blood system typically occur when a pregnancy is
at risk increasing the number of fetal cells present in the
maternal blood. A peripheral blood sample can be obtained from a
pregnant woman at a selected gestational age and the number of
fetal cells present in the sample can be detected. This detected
number of fetal cells can be compared to a known standard
representative of the number of cells present at the selected
gestational age during a normal pregnancy. The standard can be
established by taking peripheral blood samples from a group of
women at the selected gestational age believed to be having normal
pregnancies.
[0012] Other aspects of this invention relate to methods of
enriching the peripheral maternal blood sample and kits containing
reagents used to conduct the described methods. These aspects are
described in more detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic representation of the method of the
present invention by which fetal nucleated cells are isolated from
maternal cells and DNA within the fetal cells is assessed for the
occurrence of a particular fetal DNA sequence.
[0014] FIG. 2 is an autoradiograph of diluted male DNA amplified
for 222 bp sequence. Lane 1: reagent control; lane 2: .phi.X174
molecular weight standard; lane 3: 100 ng; lane 4: 10 ng; lane 5: 1
ng; lane 6: 200 pcg; lane 7: 10 pcg; lane 8: 1 pcg.
[0015] FIG. 3 is a composite autoradiograph of amplified patient
DNA. Lane 1: 10 ng normal male; lane 2: 10 ng normal female; lane
3: reagent control; lane 4: .phi.X174; lane 5: sorted cells from
patient 1 (male fetus); lane 6: sorted cells from patient 2 (male
fetus); lane 7: sorted cells from patient 3 (female fetus); lane 8:
sorted cells from patient 6 (female fetus); lane 9: sorted cells
from patient 7 (male fetus); lane 10: sorted cells from patient 8
(male fetus); lane 11: sorted cells from patient 9 (female fetus);
lane 12: cord blood from female infant whose cells were prenatally
sorted in lane 8.
[0016] FIG. 4 is a diagram demonstrating the detection of Y
chromosomal DNA sequences at various points of gestation in women
bearing male pregnancies.
[0017] FIGS. 5A, 5B, 5C, 5D, 5E and 5F are a series of histograms
obtained when FITC-anti transferrin receptor was used to determine
the presence of mononuclear cells in samples from non-pregnant
females to which male cells have been added.
[0018] FIG. 6 is a composite autoradiograph of amplified male DNA
detected in TfR cells when 10.sup.2-10.sup.6 male cells are added
to samples from non-pregnant females and in TfR.sup.- cells when
10.sup.5-10.sup.6 male cells are added to samples, from
non-pregnant females.
[0019] FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G and 7H are a series of
histograms obtained when anti HPCA-1 antibody was used to determine
the presence of mononuclear cells in samples from nonpregnant
females to which male cells have been added.
[0020] FIG. 8 is a photograph illustrating a fluorescent cell due
to the positive results of in situ hybridization of the pDP97 probe
for the Y chromosome to a fetal nucleated red blood cell.
[0021] FIG. 9 is a two-dimensional bivariate histogram depicting
forward angle light scatter on the X axis (an indication of cell
volume) and fluorescence intensity on the Y axis (a measure of
cells binding the TfR antibody. The TfR.sup.+ cells respresenting
1.3% of the maternal mononuclear cells are encased in a box.
[0022] FIG. 10 is a photomicrograph of a flow-sorted interphase
fetal nucleus isolated from maternal blood. The single black dot
represents hybridization to the Y chromosome and the three white
dots represent hybridization to the chromosome 21s. The karotype of
this nucleus is 47, XY, +21.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention relates to an in vitro method of
separating or isolating fetal nucleated cells present in the blood
of a pregnant woman (a maternal blood sample) from the pregnant
woman's cells and of separating or isolating fetal DNA from
maternal DNA. It further related to an in vitro method of prenatal
detection and/or quantitation of selected fetal DNA in fetal DNA
isolated from the maternal blood sample. The method provides a
noninvasive approach to detect and/or quantitate fetal DNA, such as
that associated with a disease or a condition whose assessment
during gestation is desired. It also provides a noninvasive means
by which the sex of a fetus can be determined.
[0024] In the present method, fetal nucleated cells are isolated
from a maternal blood sample by means of a detectable material
which binds to the fetal nucleated cells but not to maternal cells
and is then separated from the maternal sample, resulting in
separation of the fetal nucleated cells from the sample. The fetal
nucleated cells can be any undifferentiated hematopoietic cell and,
particularly, fetal nucleated erythrocytes. In one embodiment of
the present method of isolation, at least one detectably labelled
monoclonal antibody specific for an antigen present on fetal
nucleated cells, but not for an antigen present on maternal cells,
is combined with a maternal blood sample and, once bound to fetal
nucleated cells, is separated from the maternal sample.
Alternatively, at least one detectably labelled monoclonal antibody
specific for an antigen present on maternal cells, but not for an
antigen present on fetal nucleated cells is used. In a further
embodiment, the two types of monoclonal antibodies are used.
[0025] In the case in which the detectable label is a fluorescent
molecule, separation is carried out by means of flow cytometry, in
which fluorescently-labelled molecules are separated from
unlabelled molecules. This results in separation of fetal nucleated
cells, such as fetal nucleated erythrocytes, from maternal cells
and, thus, of fetal DNA from maternal DNA. That this separation has
occurred can be verified using known techniques, such as microscopy
or detection of fetal hemoglobin.
[0026] In one embodiment of the method of the present invention by
which the occurrence of a selected DNA sequence or sequences
(gene(s) or gene portion(s)) in fetal DNA is determined (detected
and/or quantitated), the isolated fetal nucleated cells, such as
fetal nucleated erythrocytes, are treated to render DNA present in
them available for amplification. Amplification of DNA from fetal
nucleated cells (fetal DNA) is carried out using a known
amplification technique, such as the polymerase chain reaction
(PCR). Amplified fetal nucleated cell DNA is subsequently separated
on the basis of size (e.g., by gel electrophoresis) and contacted
with a selected labelled probe, such as labelled DNA complementary
to a selected DNA sequence (e.g., complementary to an abnormal gene
or gene portion, or Y-specific DNA). Detection of the labelled
probe after it has hybridized to fetal DNA results in detection of
the sequence of interest in the fetal DNA. Quantitation of the
hybridized labelled probe results in quantitation of the fetal
DNA.
[0027] In a second embodiment of the present method of determining
the occurrence of a selected DNA sequence (or sequences), cells
isolated as described above are sorted onto a solid support, such
as a slide, and screened for chromosomal abnormalities using in
situ hybridization. In this embodiment, a selected nucleic acid
probe, such as a labelled DNA probe for chromosomal DNA associated
with a congenital abnormality, is combined with the fetal DNA,
under conditions appropriate for hybridization of complementary
sequences to occur. Detection and/or quantitation of the labelled
probe after hybridization results in detection and/or quantitation
of the fetal DNA to which the probe has hybridized.
[0028] The following is a description of the basis for the subject
method; of the present method of isolating nucleated fetal cells
present in the blood of a pregnant woman from maternal cells and,
subsequently, separating fetal DNA from maternal DNA; and of the
present method of prenatal determination of the occurrence
(presence/absence or quantitation) of selected DNA in fetal
cells.
[0029] Potential Sources of Fetal Genes
[0030] It has been determined that several types of fetal cells
present in the blood of a pregnant woman are a source of fetal
genes. In particular, it has been shown that fetal cells, such as
nucleated erythrocytes (also referred to as fetal NRBC), and other
undifferentiated hematopoietic precursor cells, such as
erythroblasts, lymphoblasts and myeloblasts, can be isolated or
separated from maternal blood. Fetal nucleated erythrocytes were
particularly selected for sorting based on the following
rationale:
[0031] 1. In any given fetomaternal hemorrhage, no matter how
small, the ratio of fetal erythrocytes to fetal lymphocytes should
remain the same as in whole fetal blood; thus, there would be 1,000
times as many red cells as white cells available for analysis.
[0032] 2. Normal pregnant females do not usually have circulating
NRBC; therefore, an isolated NRBC would a priori have a greater
chance of being fetal in origin.
[0033] 3. The majority of pregnancies are blood group compatible,
which means that the "transfused" NRBC would probably be tolerated
by the mother and remain in her circulation.
[0034] 4. Because they are nucleated, the NRBC contain a full
complement of fetal genes.
[0035] It has been shown that fetal nucleated erythrocytes, as well
as other types of fetal cells, can be isolated or separated from
maternal blood and that DNA present in the isolated fetal cells can
be used to assess fetal characteristics (e.g., sex, presence or
absence of chromosomal abnormalities).
[0036] Advances in Molecular Biology Applied to Fetal Cell
Sorting
[0037] Recent advances in molecular biology have had an enormous
impact on the feasibility of fetal cell identification. For
example, fluorescent in situ hybridization can be used for this
purpose.
[0038] The development of the polymerase chain reaction (PCR)
(Mullis, K., et al., Cold Spring Harb. Symp. Quant. Biol.,
51:263-272 (1986), with its capacity for DNA analysis from a single
cell (Li, H., et al., Nature. 355:414-417 (1988); Handyside, A. H.,
et al., Lancet 1:347-349 (1989)), has eliminated the technical
problems associated with the small number of fetal cells in
maternal blood. It makes DNA diagnosis from a single cell
possible.
[0039] As described below, fetal nucleated erythroblasts have been
shown to be present in blood obtained from pregnant women, thus
making maternal blood a useful/reliable potential source of fetal
DNA; fetal nucleated cells have been distinguished from maternal
cells on the basis of surface antigenic characteristics, thus
making it possible to separate the two cell types from one another;
and fetal DNA present in the separated fetal nucleated cells has
been analyzed and characterized.
[0040] Detection of Fetal Gene Sequences in Maternal Blood
[0041] One of the first steps in developing the present method of
isolating fetal nucleated cells from the maternal blood supply was
identification of monoclonal antibodies that permit identification
and separation of fetal cells from maternal cells present in blood
obtained from a pregnant woman. This has been done, as described in
detail in the Examples. As a result, it has been determined that
monoclonal antibodies which recognize maternal leucocytes and
monoclonal antibodies which recognize fetal cell surface antigens
are useful in separating maternal and fetal cells. The following is
a brief description of monoclonal antibodies which have been shown
to be useful in separating fetal nucleated cells from maternal
cells present in a maternal blood sample. However, other monoclonal
antibodies which distinguish between fetal and maternal cells on
the basis of surface antigenic differences, can also be used in the
present method.
[0042] The present method requires the use of at least one type of
antibody which is specific for (or recognizes) a surface antigen
present on fetal nucleated cells, for a surface antigen present on
maternal cells, but not specific for both. That is, the present
method can be carried out using one or more antibody which
distinguishes fetal nucleated cells from maternal cells. The
present method can be carried out using whole blood or blood
treated or processed to enrich for (increase the concentration of)
fetal nucleated cells.
[0043] Described below is the selection and successful use of
monoclonal antibodies which distinguish fetal nucleated
erythrocytes from maternal cells. It is to be understood, however,
that in a similar manner, monoclonal antibodies which make it
possible to select for another fetal nucleated cell type (or types)
can be identified and used in the present method to separate fetal
nucleated cell types from maternal cells (and, thus, fetal DNA
sources from maternal DNA).
[0044] Initial efforts focused on the elimination of contaminating
maternal leucocytes in the mononuclear cell layer and
identification of monoclonal antibodies effective in carrying out
this separation, which results in production of a maternal sample
enriched in fetal nucleated cells.
[0045] HLe-1 (Becton-Dickinson Monoclonal center, Mountain View,
Calif., catalog #7463) is a monoclonal antibody available as a
direct fluorescein isothiocyanate (FITC) conjugate. It recognizes
an antigen present on mature human leucocytes and on very immature
erythrocyte precursors, but not on mature nucleated erythrocytes
(Loken, M. E., et al., Blood, 69:255-263 (1987)). Thus, maternal
leucocytes are recognized and bound, but fetal nucleated
erythrocytes are not, making separation of the two possible. As
described in detail in Example 1, this labelled antibody was used
to eliminate maternal leucocytes in the mononuclear cell layer.
[0046] As is also described (Example 1), a combination of
monoclonal antibodies has been used for the same purpose (i.e.,
elimination of maternal cells from the blood sample). As described,
anti-monocyte antibody (M3) and anti-lymphocytes antibody (L4) have
been used to remove maternal cells from the mononuclear cell layer
resulting from density gradient centrifugation.
[0047] Monoclonal antibodies which recognize fetal nucleated cells
but do not recognize maternal cells were also identified. As
described in detail in Example 1, a monoclonal antibody which
recognizes the transferrin receptor was identified. Erythroblasts
have been shown to express the transferrin receptor (Loken, M. R.,
et al., Blood, 69:255-263 (1987)) antigen on their cell surfaces
from the BFU-E stage until nuclear extrusion (Loken, M. R. et al.,
Blood, 69:255-263 (1987)). The transferrin receptor is also present
on activated lymphocytes (Trowbridge, I. S. and M. B. Omary, Proc.
Natl. Acad. Sci. USA, 78:3039-3043 (1981)), certain tumor cells
(Greaves, M. et al., Int. J. Immunopharmac., 3:283-300 (1981)), and
trophoblast cells (Galbraith, G. M. P. et al, Blood, 55:240-242
(1980)). Thus, such an antibody is specific for or recognizes
(binds to) fetal nucleated cells, but not maternal leucocytes. As
described in Example 1, commercially available
fluorescein-conjugated monoclonal antibodies against the
transferrin receptor (TfR) were used to separate fetal nucleated
erythrocytes from maternal cells. Although the antibody is not
specific for fetal nucleated erythrocytes, it facilitated their
enrichment in the flow-sorted samples. Other monoclonal antibodies
which are able to distinguish between fetal nucleated cells and
maternal cells present in a blood sample can also be used. Such
antibodies include commercially available monoclonal antibodies and
those which can be produced using known techniques.
[0048] Separation of fetal nucleated cells from a maternal blood
sample using antibodies described above can be carried out with
samples of whole blood or a fraction of whole blood (i.e., one
resulting from treatment or processing of whole blood to increase
the proportion of fetal nucleated cells present), referred to as an
enriched maternal sample. An enriched maternal sample is produced,
for example, in a two-step process. The maternal sample is
subjected to initial separation on the basis of size, such as by
Ficoll-Hypaque density gradient centrifugation. This results in
production of a supernatant layer, which contains platelets; a
mononuclear cell layer; and an agglutinated pellet which contains
non-nucleated erythrocytes and granulocytes. The mononuclear layer
is separated from the other layers, to produce a maternal sample
which is enriched in fetal nucleated cells.
[0049] The maternal sample, whether maternal whole blood or an
enriched maternal sample, is subjected to separation, based on
surface antigenic differences between fetal nucleated cells and
maternal cells using antibodies described above. The maternal
sample is contacted with at least one monoclonal antibody which is
specific for either fetal nucleated cells or maternal cells, but
not for both and, thus, makes it possible to separate the two types
of cells. The maternal sample can be combined with a set of two or
more monoclonal antibodies, each of which is specific for either
fetal or maternal cells, but not for both. The combination of
monoclonal antibodies can be designed to enhance separation of the
two types of cells (e.g., the combination of anti-TfR antibody and
HLe-1 antibody described previously) beyond that possible with a
single monoclonal antibody. Separation of the fetal cells is
carried out using known techniques, such as flow cytometry, use of
immunomagnetic beads and cell panning. In general, the monoclonal
antibodies have a detectable label (e.g., radioactive material,
fluorophore).
[0050] In some cases, fetal nucleated cells persisting from a
previous pregnancy may be present in the peripheral blood sample.
Steps may be taken to eliminate or significantly reduce the number
of such residual nucleated cells if found to be present.
Alternatively, a type of fetal nucleated cell known to be
associated with the present pregnancy may be selected for detection
obviating any potential interference or contamination of the
detection method by the residual fetal nucleated cells. For
example, fetal nucleated cell type having a relatively short life
span may be selected for detection ensuring the fetal nucleated
cell is not from a previous pregnancy. Fetal nucleated erythrocytes
typically have a life span of about three months in the maternal
circulation. The above-identified steps can be conducted using
monoclonal antibodies which recognize antigens on the respective
cells.
[0051] An embodiment of the method of the present invention by
which fetal cells are isolated and fetal DNA is detected is
represented schematically in FIG. 1. A maternal blood sample
(typically 20 ml.) is obtained, using known techniques. The sample
is separated into component layers on the basis of size and the
mononuclear cell layer, referred to as the maternal sample enriched
in nucleated cells (or enriched maternal sample), is removed for
further processing. The enriched maternal sample is contacted with
at least one monoclonal antibody, as described above, and the
resulting fetal nucleated cell/antibody complexes are separated
using known methods (e.g., flow cytometry, immunomagnetic beads,
cell panning). Fetal DNA is crudely extracted from the resulting
complexes (e.g., by heat), thus rendering it available for
hybridization with nucleic acid probes. Fetal DNA can be analyzed
for a selected DNA sequence or DNA sequences, using known
techniques. Prior to analysis, fetal DNA can be amplified, as
needed, using known methods (e.g., PCR).
[0052] If amplification is to be carried out, the sorted samples
are amplified for an appropriate number of cycles of denaturation
and annealing (e.g., approximately 24-60). Control samples include
a tube without added DNA to monitor for false positive
amplification. With proper modification of PCR conditions, more
than one separate fetal gene can be amplified simultaneously. This
technique, known as "multiplex" amplification, has been used with
six sets of primers in the diagnosis of DMD (Chamberlain, J. S., et
al., Prenat. Diagnosis, 9:349-355 (1989)). When amplification is
carried out, the resulting amplification product is a mixture which
contains amplified fetal DNA of interest (i.e., the DNA whose
occurrence is to be detected and/or quantitated) and other DNA
sequences. The amplified fetal DNA of interest and other DNA
sequences are separated, using known techniques. Subsequent
analysis of amplified DNA can be carried out using known
techniques, such as: digestion with restriction endonuclease,
ultraviolet light visualization of ethidium bromide stained agarose
gels, DNA sequencing, or hybridization with allele specific
oligonucleotide probes (Saiki, R. K., et al, Am. J. Hum. Genet., 43
(Suppl):A35 (1988)). Such analysis will determine whether
polymorphic differences exist between the amplified "maternal" and
"fetal" samples. In one embodiment, the amplification mixture is
separated on the basis of size and the resulting size-separated
fetal DNA is contacted with an appropriate selected DNA probe or
probes (DNA sufficiently complementary to the fetal DNA of interest
that it hybridizes to the fetal DNA of interest under the
conditions used). Generally, the DNA probes are labelled (e.g.,
with a radioactive material, a fluorophore or other detectable
material). After the size-separated fetal DNA and the selected DNA
probes have been maintained for sufficient time under appropriate
conditions for hybridization of complementary DNA sequences to
occur, resulting in production of fetal DNA/DNA probe complexes,
detection of the complexes is carried out using known methods. For
example, if the probe is labelled, fetal DNA/labelled DNA probe
complex is detected and/or quantitated (e.g., by autoradiography,
detection of the fluorescent label). The quantity of labelled
complex (and, thus, of fetal DNA) can be determined by comparison
with a standard curve (i.e., a predetermined relationship between
quantity of label detected and a given reading).
[0053] The present method has been used to identify Y-specific DNA
in nucleated erythrocytes obtained from peripheral blood of
pregnant women. This is described in Example 4. Briefly, candidate
fetal cells from blood samples obtained from 19 pregnant women were
isolated by flow sorting. The DNA in these cells was amplified for
a 222 base pair (bp) sequence present on the short arm of the Y
chromosome as proof that the cells were derived from the fetus. The
amplified DNA was compared with standardized DNA concentrations;
0.1 to 1 ng fetal DNA was obtained in the 20 ml maternal samples.
In 7/19 cases, a 222 bp band of amplified DNA was detected,
consistent with the presence of male DNA in the isolated cells; 6/7
of these were confirmed as male pregnancies by karyotyping
amniocytes. In the case of the female fetus, DNA prepared from cord
blood at delivery also showed the presence of the Y chromosomal
sequence. In 10/12 cases where the 222 bp band was absent, the
fetuses were female. Thus, the Y chromosmal sequence was
successfully detected in 75% of the male-bearing pregnancies,
demonstrating for the first time that it is possible to isolate
fetal gene sequences from maternal blood.
[0054] As described in Example 6, male (Y-specific) DNA has been
detected in cells sorted from pregnant women at various points in
gestation. Briefly, the mononuclear cell layer was isolated from
venous blood samples obtained from women between 11 and 16 weeks
gestation. Separation was carried out using Ficoll/Hypaque density
centrifugation, followed by incubation with monoclonal antibodies
(Anti-TfR, anti-Leu 4 and anti-Leu.sup.M3) conjugated with a
fluorescent marker or compound (fluorescein, phycoerythrin) and
dual color analysis and flow sorting on a fluorescence-activated
cell sorter. The cells that displayed green fluorescence, but not
red fluorescence (TfR positive, Leu negative, Leu M3 negative),
were fetal nucleated cells and were separated from the remainder of
the sample. These cells were lysed, after which the DNA was
amplified and probed for the presence of a 397 bp sequence of the Y
chromosome.
[0055] The results presented in Example 6 indicate the procedure
allows the detection of the 397 bp sequence present in as little as
5 pg of male DNA. In addition, they suggest that there is a
relationship between gestational age and detection of male DNA, as
illustrated in FIG. 4. This data suggests there may be a biologic
"window" for transfer of fetal nucleated erythrocytes into maternal
circulation.
[0056] The present method also has been used to distinguish female
fetal DNA from maternal DNA. The two types of female DNA were
distinguished using amplification of paternal polymorphism, as
described in detail in Example 7. Briefly, venous blood samples
were collected from women with uncomplicated pregnancies.
Separation of fetal nucleated cells was conducted using
Ficoll/Hypaque density centrifugation, followed by incubation with
monoclonal antibodies (anti-TfR, anti-Leu 4 and anti-Leu M3)
conjugated with a fluorescent marker (fluorescein, phycoerthyrin)
and dual color analysis and flow sorting on a
fluorescence-activated cell sorter. Fetal nucleated cells
identified by displaying green fluorescence (TfR positive), but not
red fluorescence (Leu-4, Leu-3 negative), were collected and lysed.
The DNA from the cells was amplified and probed for paternal
sequences of the highly polymorphic region of chromosome 17, which
allows the distinction of female fetal DNA from maternal DNA.
[0057] The results demonstrated that DNA sequences from the father
can be identified in the autosomal chromosomes of the fetus.
Consequently, the method of the present invention can be used to
separate female fetal nucleated cells, as well as male fetal
nucleated cells, from maternal blood. Thus, the method can be used
for all DNA-based diagnostic procedures currently being used in
other methods, such as amniocentesis.
[0058] Further support for the present method's capability to
identify Y-specific DNA in nucleated erythrocytes obtained from
peripheral blood of pregnant women is given by reconstruction
experiments. As described in Example 8, male cord blood was added
to blood obtained from non-pregnant females to simulate the
presence of fetal cells in maternal blood. Briefly, venous blood
samples were collected from healthy, non-pregnant women and the
mononuclear cell layers isolated by Ficoll/Hypaque density
centrifugation. Mononuclear cells from the umbilical cords of male
infants (ranging from 10.sup.2 to 10.sup.6 cells) were added to the
mononuclear cell layers of the blood of non-pregnant women. The
cord blood contains a large percentage of nucleated erythrocytes.
The results obtained from these experiments were substantially
similar to those obtained from pregnant women at various stages in
gestation. Amplified sequences from the Y chromosome, consistent
with the presence of male DNA, were detected when 10.sup.2 male
cells were added to the female cells.
[0059] Experiments have also indicated that fetal hematopoietic
stem cells, as well as nucleated erythrocytes, may be used in the
isolation and identification of Y-specific DNA. Example 10
describes the procedure in detail. Briefly, venous blood samples
are collected from pregnant women. The mononuclear cell layer is
isolated by Ficoll/Hypaque density centrifugation and incubated
with monoclonal antibodies which are conjugated with a fluorescent
marker and directed against antigens on the surface of
hematopoietic progenitor cells (see Example 11 for discussion of
antibodies). Fluorescent cells, separated by flow sorting, have
bound antibody recognizing primitive cell surface antigens, and
thus, are hematopoietic precursor cells. After the cells are lysed
by boiling, they are subjected to polymerase chain reaction (PCR)
amplification using primers selected to amplify a 397 base pair
sequence from the Y chromosome. This method was used to study
twenty-five women, eleven of whom have confirmed male pregnancies.
In eight of these eleven women (77%), male DNA was detected in
sorted cells which showed a positive response to a human progenitor
cell antigen, thus, indicating that the cells were undifferentiated
hematopoietic stem cells. This experiment confirms that fetal
hematopoietic stem cells are circulating in the mother's blood. An
additional experiment with an antibody used to detect fetal
lymphoblasts (CD10) indicated that in eight of 17 women, male fetal
DNA was detected in CD10+ cells. Other antibodies have been
identified which recognize human stem cell antigens, as detailed in
Example 11. The data demonstrates that the types of fetal cells
present vary as the pregnancy proceeds; therefore, it may be
desirable to vary the type of antibody used in cell separation,
depending on the length of gestation.
[0060] As described in Example 12, the present method has been used
to analyze venous blood from healthy pregnant women for the
presence or absence of Y chromosomal DNA sequences and has been
shown to be highly accurate in its ability to do so, as well as to
distinguish between samples from women carrying male fetuses and
women carrying female fetuses. The only possible source of Y
chromosomal DNA in maternal blood is male fetal cells (male cells
carrying an X and a Y chromosome and female cells carry two X
chromosomes). As described, two antibodies were used, alone or in
combination, for this purpose: CD36, which recognizes a cell
surface antigen on nucleated erythrocytes and monocytes and
glycophorin A, which recognizes an antigen present on erythrocytes.
The antibodies were conjugated, directly or indirectly, to a
fluorescent dye. Fluorescent cells in the venous sample which bound
one or both antibodies were flow sorted and later amplified for Y
chromosomal sequences using PCR. Of the 18 women studied, 11 had
male fetuses and 7 had female fetuses. Y chromosomal DNA sequences
were detected in cells sorted with CD36 and/or glycophorin A
antibodies in 10 of the 11 (91%) women with male fetuses and in
none of the 7 women bearing females. These results demonstrate that
these two antibodies are particularly effective in identifying
fetal nucleated cells in maternal blood.
[0061] The results of the work described above and in the Examples
demonstrate that nucleated fetal cells have been isolated from
maternal blood; genomic DNA has been extracted from the fetal cells
and identified as being of fetal origin; fetal genes have been
amplified using PCR; and selected DNA sequences have been
identified in the fetal DNA. It demonstrates for the first time
that fetal DNA has been detected in cells isolated from maternal
blood.
[0062] Uses of the Present Method of Fetal Nucleated Cell Isolation
and Fetal DNA Characterization
[0063] Thus, it has been demonstrated that fetal DNA can be
obtained from fetal nucleated cells present in a maternal blood
sample. The method of detecting and/or quantitating fetal DNA which
is represented in FIG. 1 is useful as a tool for prenatal
assessment (e.g., as a means for assessing chromosomal
abnormalities, for determining whether DNA associated with a
disease is present, or for detecting Y-specific DNA). It is
particularly useful because it is noninvasive and requires only a
small sample of blood.
[0064] Fetal DNA sequences in fetal nucleated erythrocytes,
isolated as described herein or by other means by which fetal
nucleated cells can be separated from a maternal blood sample, can
be analyzed or assessed for the occurrence of a DNA sequence or DNA
sequences (gene(s) or gene portion(s)) which are of interest for
diagnostic or other purposes. The DNA sequence(s) or gene(s)/gene
portion(s) present in fetal cells are referred to herein as fetal
DNA of interest. For example, the selected DNA whose presence or
absence is to be determined and whose quantity can also be
determined is the gene for a disease, such as cystic fibrosis,
where the causative gene or gene portion has been cloned and
sequenced; alternatively, it is a probe for X- or Y-specific DNA.
The same procedure can also be used, with appropriate modifications
(e.g., an appropriate DNA probe, time, temperature), to detect
other genes or gene portions.
[0065] As used in a diagnostic context, such as to detect the gene
known to cause cystic fibrosis, the present method is carried out
as follows: Initially, a maternal blood sample (typically 20 ml.)
is obtained and separated into component layers based on relative
weights (e.g., by Ficoll-Hypaque density gradient centrifugation)
to remove non-nucleated erythrocytes and produce a mononuclear cell
layer. This results in production of a maternal blood sample
enriched in fetal nucleated erythrocytes. The mononuclear cell
layer is stained with at least one appropriate monoclonal antibody
(e.g., one which is specific for the type of fetal nucleated cell
to be separated from the sample). For example, a monoclonal
antibody specific for fetal nucleated cells, such as anti-TfR
antibody, described above, can be used. In general, the monoclonal
antibody used bears a detectable label. Alternatively, a
combination of selected labelled monoclonal antibodies, such as
monoclonal antibodies specific for fetal nucleated cells (e.g.,
anti-TfR antibody) and monoclonal antibodies specific for maternal
leucocytes (Hle-1 or L4 and M3), each labelled with a different
fluorescent compound, can be used to remove essentially all
maternal cells. Labelled cells are subsequently separated from one
another using a known method, such as flow cytometry. Binding of
the monoclonal antibodies to cells for which they are specific
results in production of labelled monoclonal antibody-cell
complexes. For example, in the case in which anti-TfR antibodies
and HLe-1 are used, fetal nucleated erythrocytes are bound by
anti-TfR antibody, to produce fetal nucleated erythrocytes/anti-TfR
antibody complexes, and maternal leucocytes are bound by HLe-1
antibody complexes. The fetal nucleated erythrocyte/anti-TfR
antibody complexes are separated from maternal cell/HLe-1 antibody
complexes, using, for example, flow cytometry. The fetal cells are
lysed, to produce crudely extracted fetal DNA which is subsequently
amplified, using, for example, PCR. This results in production of
amplified fetal DNA, which is subsequently separated on the basis
of size. Size-separated fetal DNA is contacted with labelled DNA
probes (i.e., in prenatal detection of cystic fibrosis, a labelled
DNA probe complementary to the gene associated with cystic
fibrosis). If the fetal DNA contains DNA of interest (in this case,
the gene associated with cystic fibrosis), fetal DNA of
interest/labelled probe complexes are formed.
[0066] Fetal DNA of interest/labelled probe complexes are
subsequently detected, using a known technique, such as
autoradiography. Simple presence or absence of labelled fetal DNA
of interest can be determined or the quantity of fetal DNA of
interest can be determined. In either case, the result is
assessment of fetal DNA obtained from a maternal blood sample for
selected DNA.
[0067] The occurrence of fetal DNA associated with diseases or
conditions other than cystic fibrosis can also be detected and/or
quantitated by the present method. In each case, an appropriate
probe is used to detect the sequence of interest. For example,
sequences from probes St14 (Oberle, I., et al., New Engl. J. Med.,
312:682-686 (1985)), 49a (Guerin, P., et al., Nucleic Acids Res.,
16:7759 (1988)), KM-19 (Gasparini, P., et al., Prenat. Diagnosis,
9:349-355 (1989)), or the deletion-prone exons for the Duchenne
muscular dystrophy (DMD) gene (Chamberlain, J. S., et al., Nucleic
Acids Res., 16:11141-11156 (1988)) are used as probes. St14 is a
highly polymorphic sequence isolated from the long arm of the X
chromosome that has potential usefulness in distinguishing female
DNA from maternal DNA. It maps near the gene for Factor VIII:C and,
thus, may also be utilized for prenatal diagnosis of Hemophilia A.
Primers corresponding to sequences flanking the six most commonly
deleted exons in the DMD gene, which have been successfully used to
diagnose DMD by PCR, can also be used (Chamberlain, J. S. et al.,
Nucleic Acids Res., 16:11141-11156 (1988)). Other conditions which
can be diagnosed by the present method include .beta.-thalassemia
(Cai, S-P., et. al., Blood, 73:372-374 (1989); Cai, S-P., et al.,
Am. J. Hum. Genet., 45:112-114 (1989); Saiki, R. K., et al., New
Engl. J. Med., 319:537-541 (1988)), sickle cell anemia (Saiki, R.
K., et al., New Engl. J. Med., 319:537-541 (1988)), phenylketonuria
(DiLella, A. G., et al., Lancet, 1:497-499 (1988)) and Gaucher
disease (Theophilus, B., et al., Am. J. Hum. Genet., 45:212-215
(1989)). An appropriate probe (or probes) is available for use in
the present method for assessing each condition.
[0068] It is also possible to separate fetal cells from maternal
cells by means other than flow cytometry, as mentioned previously,
and to analyze fetal nucleated erythrocyte DNA obtained in this
way. Such separation procedures may be used in conjunction with or
independent of flow cytometry. This is advantageous because lack of
access to a flow cytometer, as well as expense, could limit
potential applications of this technique. Thus, other methods of
fetal cell separation can be used. The separation method used can
result in elimination of unwanted cells ("negative selection") or
isolation of rare but desirable cells ("positive selection").
[0069] In one embodiment, the maternal cells are depleted prior to
fetal cell sorting. The mononuclear cell layer is initially
isolated from the blood of pregnant women by Ficoll-Hypaque
centrifugation. The resulting cell suspension consists
predominantly of maternal cells; in order to enrich the eventual
proportion of fetal cells present, the maternal cells are
selectively removed by incubating the cells with antibodies
attached to a solid support. Such supports include magnetic beads,
plastic flasks, plastic dishes and columns. The antibodies bind
antigens present on the cell surface of mature leukocytes. Thus,
the majority of maternal leukocytes are eliminated by virtue of
being bound to the solid support. The total number of cells
remaining in the cell suspension is smaller, but the proportion of
fetal cells present is larger.
[0070] Separation by immunomagnetic beads or by cell panning can
also be used. In this embodiment, the mononuclear cell layer is
isolated, as described previously. This layer is then mixed with
antibody-coated polymer particles containing magnetic cores (e.g.,
"Dynabeads"). These immunomagnetic beads are available coated with
a variety of antibodies. For example, immunomagnetic beads coated
with antibody to leucocyte antigens and antibody to mouse
immunoglobulins, which can be subsequently conjugated to mouse
monoclonal antibody against the human transferrin receptor, can be
used. After mixing, the rosetted cells are isolated with a magnetic
particle concentrator. In one embodiment, two sets of
antibody-coated immunomagnetic beads are used in succession. First,
the maternal leucocytes are depleted and then the remaining TfR
positive cells are collected. Subsequent steps in the method
(amplification, separation, contact with an appropriate DNA probe
or probe set) are as described for cells separated by flow
cytometry.
[0071] Mueller et al. (Lancet, 336:197-200 (1990)) have described a
method of isolating placenta-derived trophoblast cells in the blood
of pregnant women using magnetic beads. This method included mixing
1 ml of monoclonal antibody hybridoma culture supernatant with
2.times.10.sup.7 magnetic beads precoated with sheep antibody to
mouse IgG (Fc fragment) (Dynabeads M-450, Dynal AS, Oslo, Norway),
and incubated overnight at room temperature. The coated beads were
stored at 4.degree. C. and washed three times in ice-cold RPMI 1640
medium containing lithium heparin (10 IU/ml). The blood from the
pregnant women was collected into tubes containing 10 IU of lithium
perml of whole blood, diluted 1:10 with RPMI containing lithium,
and incubated with the antibody coated beads at 4.degree. C.
overnight. The desired cells were bound to the antibody on the
bead; the beads collected by means of a cobalt-samarium magnet.
Although in this case the antibody was directed against trophoblast
antigens a similar technique can be utilized with, for example,
antibody to cell surface antigens present on fetal nucleated
erythrocytes and not present on maternal cells. An advantage to
this particular technique is that an initial step which results in
mononuclear cell isolation is not added. Additionally, the magnetic
beads can be used for both positive (fetal cells) and negative
(maternal cells) selection.
[0072] An alternative method of isolation can be a modification of
the method described by R. J. Berenson et al. (J. of Immunol.
Methods, 91(1986)) in which the high affinity between the protein
avidin and the vitamin biotin was exploited to create an indirect
immuno-adsorptive procedure. In this technique, avidin was linked
to cyanogen bromide activated sepahrose 6MB beads and washed in an
alternating fashion with coupling buffer (0.1 M NaHCO.sub.3 in 0.5
M NaCl at pH 8.3). and washing buffer (0.1 M sodium acetate in 0.5
M NaCl at pH 4.5) and stored at 4.degree. C. The blood cells were
incubated with 1) murine monoclonal antibody, and 2) biotinylated
goat anti-mouse immunoglobulin. A 3 ml column of gel was packed in
Pharmacia K 19/15 column. The treated cells were passed through the
column in phosphate buffered saline containing 2% bovine serum
albumin. Adherent cells were dislodged by mechanical agitation.
This technique can be applied to fetal cell separation if the
antibodies used recognize fetal cell surface antigens or maternal
cell surface antigens, but not both. Variation in methods for
conjugating antibodies to beads exist; examples include those
described by Thomas and co-workers (Thomas, T. E., et al. (J. of
Immuno. Methods, 120:221-131 (1989)) and by deKretser and
co-workers (deKretser, T. A., et al. (Tissue Antigens, 16:317-325
(1980)). The use of an antibody-bound column does not require the
preliminary isolation of the mononuclear cell fraction from whole
blood.
[0073] Another alternative to mononuclear cell isolation is to
selectively lyse maternal non-nucleated erythrocytes. A number of
buffers, including 0.17M NH.sub.4Cl, 0.01M Tris, pH 7.3, have been
described in the literature and are well known in isolation of
hematopoietic stem cells for bone marrow transplantation. Other
buffers ("Lyse and Fix", GenTrak) are available commercially.
[0074] Once the fetal cells are isolated from maternal blood, they
may be cultured to increase the numbers of cells available for
diagnosis, if desired. E. Fibach et al. (Blood, 73:100-103 (1989))
have described a method that supports the growth of human
hematopoietic progenitor cells. This step-wise method involves 1)
initial culture in the presence of conditioned medium from human
bladder carcinoma cells, 2) removal of leucocytes by harvest of
non-adherent cells and lysis with monoclonal antibodies and 3)
reculture of cells in medium supplemented by recombinant
erythropoietin.
[0075] Other methods of separating fetal nucleated cells from
maternal cells can also be used, provided that they make it
possible to differentiate between fetal cells and maternal cells,
and to isolate one from the other.
[0076] A kit for use in carrying out the present method of
isolating and detecting fetal DNA of interest, such as a
chromosomal abnormality associated with a disease or other
condition, in a maternal blood sample can be produced. It includes,
for example, a container for holding the reagents needed; the
reagents and, optionally, a solid support for use in separating
fetal nucleated cell/specific antibody complexes from other sample
components or for removing maternal cells complexed with a specific
antibody. For example, reagents in a kit to be used in detecting
fetal DNA of interest after amplification of fetal DNA by PCR can
include: 1) at least one antibody specific for a surface antigen
characteristic of fetal nucleated cells but not specific for a
surface antigen characteristic of maternal leucocytes; selected DNA
primers for use in amplifying fetal DNA by PCR; and at least one
DNA probe complementary to the fetal DNA to be detected (fetal DNA
of interest). The kit, as indicated, can also include a solid
support to be used in separating complexes formed from other
samples components. Such solid support can be, for example, a glass
slide, nitrocellulose filter, or immunomagnetic beads and can have
affixed thereto an antibody selective for the antibody present in
the fetal nucleated cell/specific antibody complexes.
[0077] The present invention will now be illustrated by the
following examples, which are not intended to be limiting in any
way.
EXAMPLE 1
Antibody Selection for Isolation and Sorting of Fetal Nucleated
Erythrocytes (NRBCs)
[0078] Removal of Maternal Leucocytes from Maternal Blood Using
Human Leucocyte Antigen (HLe-1)
[0079] 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.
[0080] The mononuclear cell layer from peripheral blood samples in
63 pregnant women, 15 nonpregnant adults, and 39 umbilical cords,
was stained with FITC-HLe-1 for flow cytometric analysis. Umbilical
cord samples were used as a substitute for whole fetal blood.
Representative histograms displaying fluorescence versus low-angle
light scatter (an approximation of cell size) for each of the three
groups were generated. Histogram peaks were identified that
corresponded to leucocytes, erythrocytes and platelets. In 9
pregnant women, 7 nonpregnant adults and 12 umbilical cord samples,
fluorescent (HLe-1 positive) and non-fluorescent (HLe-1 NEGATIVE)
cell populations were sorted for detailed microscopy after
Wright-Giemsh staining. While the HLe-1 positive populations were
always composed of leucocytes independent of the sample source, the
HLe-1 negative populations differed.
[0081] In cord blood, the HLe-1 negative cells were nonnucleated
and nucleated erythrocytes with occasional platelets. In the
pregnant women, there were platelets, non-nucleated erythrocytes,
and a very rare NRBC. In non-pregnant adults, only platelets and
debris were seen. Thus, cord blood, with it high percentage of
NRBCS, was used as a reference to establish cell sorting
parameters. Microscopy confirmed the specificity of the
antibody-antigen binding and that the sorted HLe-1 negative cells
were relatively free from leucocyte contamination. These sorting
parameters were utilized to isolate potential fetal NRBC on 40
pregnancies.
[0082] Enrichment of Fetal NRBC in Maternal Blood Using Transferrin
Receptor Antigen (TfR)
[0083] The transferrin receptor (Newman, R., et al., Trends
Biochem. Sci. 1:397-399 (1982)) is a surface glycoprotein important
in cellular iron transport. The TfR is present on activated
lymphocytes (Trowbridge, I. S., et al., Proc. Natl. Acad. Sci. USA,
78:3039-3043 (1981)), certain tumor cells (Greaves, M. et al., Int.
J. Immunopharmac., 3:283-300 (1981)), and trophoblast cells
(Galbraith, G. M. P., et al., Blood, 55:240-242 (1980)).
Erythroblasts express the TfR on their cell surfaces from the BFU-E
stage until nuclear extrusion (Loken, M. R., et al., Blood,
69:255-263 (1987)). Thus, TfR is an excellent "candidate antigen"
for enrichment of fetal NRBCs found in maternal blood. Monoclonal
antibody against TfR is available as both a fluorescein conjugate
(Becton-Dickinson catalog #7513) and a phycoerythrin (PR) conjugate
(gift of Dr. Michael Loken, Becton-Dickinson). The mononuclear cell
layer was isolated from peripheral blood samples in 6 pregnant
women, 4 non-pregnant adults, and 3 newborn umbilical cords for TfR
analysis and microscopy. Representative histograms of fluorescence
versus light scatter from these three groups were generated.
[0084] Whereas umbilical cord samples had a large population of
fluorescent TfR positive cells) that were heterogenous in size,
non-pregnant adults and pregnant adults had smaller percentages of
fluorescent cells that clustered in discrete groups. In addition,
there were slight differences in the percentages of TfR positive
cells in the pregnant (mean=0.83) versus non-pregnant (mean=0.32)
samples studies.
[0085] Microscope studies of the TfR positive cells were performed
using Wright-Giemsa stain for morphology and Kleihauer-Betke
technique for the detection of fetal hemoglobin (Kleihauer, E., et
al., Klin Wochenschr., 35:637-638 (1957)). In the umbilical cord
samples, large numbers of nucleated and non-nucleated erythrocytes
containing fetal hemoglobin and occasional leucocytes were
identified visually. In the pregnant women, the predominant cell
types were nucleated and non-nucleated erythrocytes containing
fetal hemoglobin, although leucocytes were infrequently observed.
In contrast, the samples from the non-pregnant controls consisted
almost exclusively of lymphocytes and monocytes. Because
trophoblast cells express TfR, it was postulated that they might be
present in the sorted population from the pregnant women; none was
detected.
[0086] Dual Antibody Analysis
[0087] Because both antibodies enriched the proportion of NRBCs
present, but did not completely exclude other cell types in the
sorted samples, combinations of antibodies were used to isolate
pure populations of fetal NRBCs. Preliminary dual antibody studies
were performed using PE-conjugated TfR and FITC-conjugated HLe-1.
NRBCs are TfR positive and HLe-1 negative, whereas maternal
leucocytes are HLe-1 positive. These experiments worked well and
resulted in separation of maternal leucocytes.
[0088] Thus, the work described above defined flow cytometric
parameters for enrichment and sorting of NRBCs in peripheral blood
from pregnant women. In addition, microscopic studies revealed that
morphologic differences occur in mononuclear cell populations
derived from venous blood samples in pregnant versus non-pregnant
adults.
EXAMPLE 2
DNA Hybridization Studies in HLe-1 Negative Cells Sorted from
Maternal Blood
[0089] 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.
[0090] DNA Isolation
[0091] HLe-1 negative cells from cord blood and pregnant women were
sorted into test tubes. Conventional methods of DNA isolation as
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
[0092] 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).
[0093] 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 81/2 and 38 weeks
gestation. Results were the following:
1 Delivered Delivered Lost To Dot Blot Hybridization Male Female
Follow- with Y Chromosomal Probe Infant Infant up + 3 2 0 - 21 12
2
[0094] 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 occurred,
but that this DNA bound to the Y probe nonspecifically. Thus, the
filters interpreted as "positive" for male DNA might actually have
been "positive" for fetomaternal hemorrhage.
EXAMPLE 4
Use of the Polymerase Chain Reaction (PCR) to Amplify Gene
Sequences in Sorted Fetal Cells
[0095] PCR, which has a capacity for making 10.sup.6 copies of rare
target gene sequences, was used to amplify gene sequences in sorted
fetal cells. Optimum conditions for PCR, given the minute amounts
of DNA expected after a fetal cell sort (approximately 1 pg to 100
ng), were determined. Experimental conditions were modified as new
information became available. For example, Taq polymerase was used
instead of Klenow fragment of E. Coli DNA polymerase (Kogan, S. C.
et al., New England J. Med. 317:990 (1987)) because of its
increased specificity in DNA replication.
[0096] Initially, studies were performed on repeated sequences from
the long arm of the Y chromosome, probe Y431-Hinfa (given by Dr.
Kirby Smith, Johns Hopkins University, Baltimore, Md.) and the
short arm of the Y chromosome, probe Y411 (Given by Dr. Ulrich
Muller, Children's Hospital, Boston, Mass.). Repeated sequences
were selected because they would create a stronger amplification
signal from a rare male fetal cell. Y411 is identical to Y156
(Muller, U., et al., Nucleic Acids Res., 14:1325-1329 (1986)), is
repeated 10-60 fold, and is absolutely Y specific on Southern
blots. Sequence Y431 has autosomal homology in females that limited
its usefulness in sex determination.
[0097] PCR Standardization
[0098] To define the minimum amount of DNA detectable in maternal
blood, a series of standardization experiments were done. DNA from
male and female individuals was prepared in tenfold dilutions (1 pg
to 1 mcg) and amplified using the standard reagents in the
GeneAmpkit (Perkin-Elmer Cetus cat #N801-0055) on a Perkin-Elmer
DNA Thermal Cycler. Primers 411-01 and 411-03 were designed to
amplify a 222 base pair (bp) sequence within probe Y411. The number
of amplification cycles varied between 18 and 30. Amplified DNA
samples were electrophoresed on agarose gels, transferred to nylon
filters, and hybridized to .sup.32P-labeled Y411 probe. While it
appeared possible to detect Y specific bands on autoradiographs in
lanes containing as little as 10 pg of male DNA, results were often
muddled by the presence of amplified DNA in female lanes or control
lanes containing no added DNA. The phenomenon of "false positive
amplification" has now received universal recognition (Lo, Y-M.D.,
et al., Lancet, 2:697 (1988); Kwok, S., et al, Nature, 339:237-238
(1989)).
[0099] Elimination of "False Positive" Amplification
[0100] Due to the limited amount of starting material in a fetal
cell sort, every effort was made to eliminate background
amplification in order to determine which fetuses truly possess Y
chromosomal DNA. Thus, measures were taken to prevent aerosol
contamination of male DNA. All PCRs were performed under sterile
conditions, wearing gloves, and using positive displacement
pipettes. All reagents were prepared in a sterile manner and
incubated overnight prior to PCR with a restriction endonuclease
having a digestion site within the target sequence. These
precautions resulted in a significant decrease and virtual absence
of false positive amplification, as monitored by running control
reactions with all reagents but no DNA.
[0101] Successful Isolation and Amplification of Fetal Gene
Sequences from NRBCs in Maternal Blood
[0102] After eliminating sources of DNA contamination and
determining that as little as 10 pg of male DNA (1 cell 7 pg of
DNA) could be detected after PCR amplification, candidate fetal
cells from the peripheral blood of 19 women at 121/2 to 17 weeks
gestation were sorted. Monoclonal antibody against TfR was used to
identify the presumed NRBC. The DNA in the sorted cells was
amplified for the 222 bp sequence in probe Y411 as proof that the
cells were derived from the fetus in male pregnancies. In 7/19
cases the 222 bp band of amplified DNA was detected on
autoradiographs, consistent with the presence of male DNA in the
isolated cells; 6/7 of these were confirmed as male pregnancies by
karyotyping amniocytes. In the case of one female fetus, repeat
studies at 32 weeks gestation and cord blood at delivery also
showed the presence of the Y chromosomal sequence. This result
might be explained by a low level of sex chromosome mosaicism,
XX/XY chimerism (Farber, C. M., et al., Hum, Genet., 82:197-198
(1989)), or the presence of the Y411 sequence in single copy on the
X chromosome or autosomes. In 10/12 cases where the 222 bp was
absent, the fetuses were female. Therefore, detection of the Y
chromosomal sequence was successful in 6/8 of 75% of the
male-bearing pregnancies. In the two pregnancies where male DNA was
not detected, there may have been fetomaternal blood group
incompatibility. Alternatively, there may not have been
fetomaternal hemorrhage or the number of NRBCs present may have
been below the limit of sensitivity for detection of DNA. The
conditions used made it possible to detect a minimum of 100 pg of
fetal DNA, or the equivalent of 15 fetal cells. The limit of
sensitivity can be improved by extending the number of cycles used
in PCR. This work demonstrated that for the first time, fetal DNA
was detected in cells isolated from maternal blood.
[0103] To further decrease false positive amplification and permit
detection of fetal DNA at the single cell level on agarose gels,
PCR is being carried out using primers derived from a single copy
of sequence specific for the long arm of the Y chromosome, PY49a
(Guerin, P., et al., Nucleic Acids Res., 16:7759 (1988)). In
preliminary experiments using 60 cycles of PCR, Y chromosomal DNA
is visible on ethidium-bromide stained agarose gels. This
extraordinary degree of sensitivity will now be applied to DNA from
sorted fetal cells.
EXAMPLE 5
Determination of the Volume, Morphology and Universality of
Fetomaternal Hemorrhage
[0104] a. General Strategy
[0105] It is also possible, because of the availability of the
present method of isolating fetal nucleated cells from blood
obtained from a pregnant woman, to determine whether fetal cells
can be found in the maternal blood in all pregnancies. A data base
can be created that can provide information on the number and type
of fetal cells circulating in maternal blood as pregnancy
progresses. Based on previous work, it is anticipated that there
will be a normal range of values that is dependent on gestational
age; deviation from these values will be studied as a potential
indication of a pregnancy at risk. Specifically, large amounts of
fetal blood in the maternal circulation may be correlated with
placental abnormalities, threatened miscarriage and intrauterine
growth retardation.
[0106] Maternal venous blood samples are collected from pregnant
women, generally prior to any invasive procedures. In general, a
single 20 ml. venous blood sample will be obtained. In a subgroup
of patients, permission will be sought to draw blood samples every
4 weeks to follow changes in numbers of fetal cells present. Blood
is collected in EDTA, diluted 1:1 with Hanks Balanced Salt Solution
(HBSS), layered over a Ficoll-Hypaque column (Pharmacia) and spun
at 1400 rpm for 40 minutes at room temperature. The mononuclear
cell layer will be isolated, washed twice with HBSS and stained
with fluorescent monoclonal antibodies. For example, this can be a
combination of fluorescein isothiocyanate-conjugat- ed
antitransferrin receptor (TfR) and phycoerythrin-conjugated
anti-monocyte antibodies (M3, Becton-Dickinson catalog 17497) and
anti-lymphocyte antibodies (L4, Becton Dickinson catalog #7347).
The staining occurs on ice, in phosphate buffered saline (PBS)
containing 2% fetal calf serum and 0.1% sodium azide. The cells are
washed in PBS prior to flow cytometry. Analysis and sorting are
performed on a Becton-Dickinson FACS-IV interfaced with a Consort
40 program. Data will be acquired on the relative size and
fluorescence (in two colors) of the analyzed cells. Cells that are
fluorescent in the green wavelength (TfR positive) and not
fluorescent in the red wavelength (L4 and M3 negative) will contain
the presumed fetal NRBCs. The percentage of these cells in the
mononuclear cell layer are recorded and analyzed as a function of
gestational age. These cells are sorted for microscopy and PCR
amplification. In addition, cells that are not fluorescent in the
green wavelength (TfR negative) but are fluorescent in the red
wavelength (L4 and/or M3 positive) are sorted as a presumed
maternal leucocyte population and source of maternal DNA
polymorphisms.
[0107] An additional benefit of studying nucleated fetal cells in
maternal blood is that the amount of fetal DNA present can be
extrapolated to determine the extent of fetomaternal hemorrhage in
normal and unusual pregnancies. In the pregnancies studied, an
average amount of 1 ng of fetal DNA (corresponding to 150 NRBCs)
was present. Using published values of the number of NRBCs per
liter of fetal blood at 16 weeks, (3.6.times.10.sup.9) (Millar, D.
S. et al., Prenat. Diagnosis, 5:367-373 (1985); (Forestier, F., et
al., Pediatr. Res., 20:342-346 (1986)) and doing simple algebra,
these results were calculated to be consistent with 2-20 .mu.l
hemorrhage of fetal blood into maternal circulation. This is a
trivial amount when compared with the fetoplacental blood volume at
16 weeks, about 20 ml. It is important to validate and extend these
results to generate normative data regarding fetomaternal
transfusion in early pregnancies. It will be equally important to
correlate deviations from the expected results with pregnancy
complications.
EXAMPLE 6
Detection of Male DNA in Cells Sorted from Pregnant Women at
Different Points in Gestation
[0108] Venous blood samples (20 ml) were collected in EDTA from
healthy women with uncomplicated pregnancies, prior to invasive
diagnostic procedures, at different points in gestation. The
mononuclear cell layer was isolated by Ficoll/Hypaque density
centrifugation and incubated with the monoclonal antibodies
fluorescein (FITC)-conjugated anti-TfR, phycoerythrin
(PE)-conjugated anti-Leu 4 and PE-conjugated anti-Leu M3
(Becton-Dickinson). Dual color analysis and flow sorting were
performed on a fluorescence-activated cell sorter.
[0109] Cells that display green fluorescence but not red
fluorescence (TfR positive, Leu 4 negative, Leu M3 negative) were
collected into sterile micro test tubes and frozen at -20.degree.
C. Prior to polymerase chain reaction amplification, the cells were
lysed by boiling. The polymerase chain reaction (PCR) was performed
under standard conditions using standard reagents as described in
Example 4. The primers used to amplify material from the Y
chromosome define a 397 base pair (bp) sequence. After PCR, the
patient samples were analyzed with conventional Southern blots
using .sup.32P labelled probe. Ethidium bromide stained agarose
gels and autoradiographs were examined for the presence of the 397
bp band, which is considered significant only if reagent controls
do not reveal false positive amplification.
[0110] Under the reaction conditions described above, it was
possible to detect the 397 bp male specific band if 5 pg of male
DNA was present. This is approximately the amount of DNA present in
one cell. When excess female DNA (500 ng) was added to the reaction
mixture, the male specific band was consistently detectable at 100
pg.
[0111] FIG. 4 represents a summation of samples obtained from
twelve women bearing male fetuses. These samples were taken at
different times in pregnancy, and one woman was sampled twice. The
data indicates that there is a relationship betwen gestational age
and the detection of male DNA. This implies a potential biologic
"window" for the transfer of fetal nucleated erythrocytes into the
maternal circulation.
EXAMPLE 7
Detection of Female Fetal DNA by Amplification of Paternal
Polymorphisms
[0112] Venous blood samples (20 ml) were collected in EDTA from
health women with uncomplicated pregnancies. The mononuclear cell
layer was isolated by Ficoll/Hypaque density centrifugation and
incubated with the monoclonal antibodies fluorescein
(FITC)-conjugated anti-TfR, phycoerythrin (PE)-conjugated anti-Leu
4 and PE-conjugated anti-Leu M3 (Becton-Dickinson). Dual color
analysis and flow sorting were performed on a
fluorescence-activated cell sorter.
[0113] Cells that display green fluorescence but not red
fluorescence (TfR positive, Leu 4 negative, Leu M3 negative) were
collected into sterile micro test tubes and frozen at -20.degree.
C. Additionally, cells that displayed red fluorescence but not
green fluorescence (TfR negative, Leu 4 positive, Leu M3 positive)
were collected in an identical manner. Prior to polymerase chain
reaction (PCR) amplification, the cells were lysed by boiling. PCR
was performed using buffers containing 1 mM MgC.sup.12. The primers
used in PCR amplify a highly polymorphic region of chromosome 17.
Amplified DNA sequences correspond to blocks of genes transmitted
directly from parent to child. As a result of the high degree of
individual variation in these sequences, it is uncommon for two
parents to manifest identical DNA patterns. Thus, it is possible to
demonstrate inheritance of the paternal sequences in the sorted
fetal cells. Since these sequences are from chromosome 17, they are
independent of fetal sex, and may be used to distinguish female
fetal DNA from maternal DNA. Amplified DNA was separated by
electrophoresis through ethidium bromide stained agarose gels. The
DNA was transferred to nylon filters and probed using .sup.32P
labeled sequence. The maternal DNA, paternal DNA TfR.sup.+ cells,
and TfR.sup.- cells were then compared.
[0114] In 5 of 10 pregnant women, it was possible to show the
presence of paternal sequences in the sorted candidate fetal cell
population. In the other 5 women, no differences were seen between
the maternal DNA and the DNA obtained from the candidate fetal
cells.
EXAMPLE 8
Reconstruction Experiments Using Non-Pregnant Female Blood and
Added Male Cord Blood To Simulate the Presence of Fetal Cells in
Maternal Blood
[0115] Venous blood samples (20 ml) were collected in EDTA from
healthy non-pregnant women. Umbilical cord blood samples (10 ml)
were collected in EDTA from normal newborns. The mononuclear cell
layer was isolated by Ficoll/Hypaque density centrifugation. Cell
counts were performed with a hemocytometer. Separate aliquots of
cells were made containing: 1) female cells alone; 2) female cells
plus 10.sup.2 added male cord blood cells; 3) 3 female cells plus
10.sup.3 added male cord blood cells; 4) female cells plus 10.sup.4
added male cord blood cells; 5) female cells plus 10.sup.5 added
male cord blood cells; 6) female cells plus 10.sup.6 added male
cord blood cells; 7) male cord blood cells alone. The separate
aliquots were then incubated with the individual monoclonal
antibodies being tested. Analysis and sorting were performed using
a flow cytometer. For each aliquot, a bivariate histogram was
obtained, and gating parameters were established for antibody
positive and antibody negative cells. The sorted cells were
collected into sterile micro test tubes and frozen at -20.degree.
C. PCR amplification was performed with primers that detect a 397
bp sequence unique to the Y chromosome. The presence of a band at
397 bp in autoradiographs was used to confirm the presence of male
umbilical cord blood cells in sorted samples.
[0116] FIG. 5 shows the histograms obtained when FITC-anti
transferrin receptor is used. In the non-pregnant female, 0.1% of
the mononuclear cells react with the antibody. In male cord blood,
24.9% of the mononuclear cells react with the antibody. With the
addition of more and more umbilical cord cells to the non-pregnant
female cells, and increased percentage of cells that react with the
antibody is seen.
[0117] FIG. 6 shows that male DNA is detected in the TfR.sup.+
cells when 102-106 male cells are added. Male DNA is detected in
the TfR.sup.- cells when 10.sup.5-10.sup.6 male cells are added.
This results from the presence of male white blood cells in the
TfR.sup.- population.
[0118] FIG. 7 shows the histograms obtained when anti HPCA-1
antibody is used. In the non-pregnant female, 0.9% of the
mononuclear cells react with antibody. In umbilical cord blood, a
well-defined population of cells is seen, but the percentage is
only 1.1%. Thus, the addition of umbilical cord blood cells to the
nonpregnant female cells is not seen on the histograms as clearly
as with the transferrin receptor antibody. An increased number of
HPCA.sup.+ cells were collected as the amounts of added cord blood
cells increased.
[0119] In agarose gels, the 397 bp band consistent with DNA was
detected in the HPCA.sup.+ cells when 10.sup.3-10.sup.5 male cells
were added to the female cells. Male DNA was detected in agarose
gels in the HPCA cells when 10.sup.6 male cells were added to the
female cells.
EXAMPLE 9
In situ Hybridization Using Molecular Probes Recognizing Individual
Chromosomes in Flow Sorted Nucleated Erythrocytes
[0120] To demonstrate diagnostic utility of the present invention,
a DNA probe set was constructed of chromosome specific probes that
provided both good signal to noise ratios and good spatial
resolution of the fluorescent signals. Accordingly, specific probes
were developed for five chromosomes frequently seen as liveborn
aneuploidies; chromosomes 13, 18, 21, X and Y. A probe for
chromosome 1 was used as a control. In constructing the probes, the
general strategy was to identify a starting clone that mapped to
the desired chromosomal region by multiple genetic and physical
methods, and then to use that clone to identify a matching cosmid
"contig" which was then used as a hybridization probe.
[0121] Hybridization of the high copy number repeat sequences was
suppressed by inclusion of total genomic human DNA, and the
chromosomal specificity verified by hybridization to metaphase
spreads. The probes gave sharp, punctate fluorescent signals in
interphase cells that were easily discriminated and enumerated. The
Y probe used in this study was pDP97, a repetitive clone (a 5.3 kb
EcoRI Y fragment from cosmid Y97 subcloned into EcoRI site of
pUC-13). All probes were labeled with biotin, hybridized under
suppression conditions, and specific hybridization detected by
conjugated strepto-avidin-FITC, which showed as a single "dot" in
the FITC image. As illustrated in FIG. 8, the Y chromosome was
detected by in situ hybridization of a pDP97 probe for the Y
chromosome in a fetal nucleated red blood cell. Thus, prenatal
diagnosis for chromosomal abnormalities could be performed on fetal
cells isolated from maternal blood.
EXAMPLE 10
Detection of Fetal Hematopoietic Stem Cells in Maternal
Circulation
[0122] Venous blood samples (20 ml) were collected in EDTA or
citrate dextrose from healthy women with uncomplicated pregnancies,
prior to invasive diagnostic procedures, at different points in
gestation. The mononuclear cell layer was isolated by
Ficoll/Hypaque density centrifugation and incubated with monoclonal
antibodies directed against antigens expressed on the cell surface
of hematopoietic progenitor cell. These antibodies are described in
Example 2. The antibodies were either directly or indirectly
conjugated to a fluorescent dye. Analysis and flow sorting of
fluorescent cells were performed on a fluorescence-activated cell
sorter. Fluroescent cells are cells that have bound antibody
recognizing primitive cell surface antigens; thus, they are
hematopoietic precursor cells. These cells were physically sorted
into sterile micro test tubes and frozen at -20.degree. C. Prior to
polymerase chain reaction amplification, the cells were lysed by
boiling. The polymerase chain reaction (PCR) was performed under
standard conditions using standard reagents. The primers selected
amplified material from the Y chromosome as a means of detecting
male fetal cells. These primers defined a 397 base pair sequence.
After PCR, the patient samples were analyzed with conventional
Southern blots using a .sup.32P labeled probe. Ethidium bromide
stained agarose gels and autoradiographs were examined for the
presence of the amplified 397 bp band, which was considered
significant only if reagent controls did not reveal false positive
amplification.
[0123] Twenty five women were studied with antibody to a human
progenitor cell antigen (HPA). This antigen has been given a
cluster of differentiation (CD) designation as CD34. Cells that are
CD34+ are undifferentiated and represent hematopoietic stem cells.
Eleven of the twenty five women whose peripheral blood was sorted
have confirmed male pregnancies. In 8/11 (72%) of those women, male
DNA was detected in the sorted CD34+ cells. This confirms that
fetal hematopoietic stem cells are circulating in mother's
blood.
[0124] Additionally, antibodies were used against the oncofetal
antigen expressed in many leukemias (CALLA) to detect fetal
lymphoblasts in the maternal circulation. This antibody is
designated as CD10. Seventeen women have been sorted with antibody
to CD10. In 8/17, evidence of male fetal DNA has been detected in
CD10+ cells.
EXAMPLE 11
Additional Antibodies to Detect Fetal Hematopoietic Cells in
Maternal Circulation
[0125] Human fetal umbilical cord blood is being used as a source
of mononuclear cells for study of fetal cell populations, in order
to determine additional antibodies to detect fetal hematopoietic
cells in material circulation. Pure fetal blood samples were tested
from as early as 19 weeks gestation.
[0126] The mononuclear cell layer is isolated as described in
example 10. Mononuclear cells are incubated with mouse monoclonal
antibodies to human stem cell antigens (CD34, CD10, CD38, which
recognizes myeloblasts, CD33, HLA-DR, CD36, glycophorin A, CD71,
8G12, THB-7 and others). Many of these antibodies are available
directly conjugated to a fluorescent dye. If the antibody is not
fluorescent, a sandwich technique is used to attach
fluorescein-conjugated goat anti mouse antibody to the primary
antibody.
[0127] Our data thus far has demonstrated that there are
differences in the types of fetal cells present as the pregnancy
proceeds. Approximately 4% of the fetal mononuclear cells are CD10+
until 34 weeks gestation. CD10+ cells are not detectable at term.
Approximately 5% of fetal mononuclear cells are CD34+. Between 8
and 18% of fetal mononuclear cells are CD38+. The percentages of
CD38+ cells increase during the pregnancy. Therefore, varying the
type of antibody used in cell separation based on the length of
gestation may help increase the isolation of fetal cells.
EXAMPLE 12
Detection of Y Chromosomal DNA Sequences in Venous Blood Samples
Obtainly Only from Women Having Male Fetuses
[0128] Venous blood samples (20 ml) were collected in EDTA or
citrate dextrose from healthy women with uncomplicated pregnancies,
prior to invasive diagnostic procedures, at different points in
gestation. The mononuclear cell layer was isolated as described in
Example 10. Alternatively, mononuclear cells could be obtained by
(nonnucleated) red cell lysis buffers. In this example, the
specific monoclonal antibodies chosen to label the fetal cells of
interest are CD36 and glycophorin A, used singly or in combination.
These antibodies are either directly or indirectly conjugated to a
fluorescent dye. CD36 recognizes a cell surface antigen present on
nucleated erythrocytes and monocytes, whereas glycophorin A
recognizes an antigen present on erythrocytes. Fluorescent cells
that have bound either or both antibodies were flow sorted; such
cells were frozen and subsequently amplified for Y chromosomal
sequences by the polymerase chain reaction as described in Example
10.
[0129] In a group of 18 women studies, 11 had male fetuses and 7
had female fetuses. Y chromosomal DNA sequences were detected in
the cells sorted with CD36 and/or glycophorin A antibodies in 10/11
(91%) of the women having males. None of the women bearing females
(0/7) had Y chromosomal DNA sequences detected. The probability of
obtaining these results by chance, p=0.00025 by Fisher exact test.
Thus, the antibodies CD36 and glycophorin A are particularly
effective in identifying fetal nucleated cells circulating in
maternal blood.
EXAMPLE 13
Detection of Fetal Cells with 47 XY +21 Karotype in Maternal
Peripheral Blood
[0130] A peripheral blood sample (20 ml.) was obtained from a
pregnant woman at nineteen weeks of gestation. A previous
ultrasound examination performed at 17 weeks gestation revealed
fetal malformations consistent with a diagnosis of Down Syndrome.
An amniocentesis was performed for fetal chromosome analysis and
results of the karotype were 47, XY, +21.
[0131] Flow Cytometry
[0132] The woman's venous blood was collected in ethylenediamine
tetraacetic acid (EDTA), diluted 1:3 with Hanks' balanced salt
solution, layered over a Ficoll/Hypaque column (Pharmacia) and spun
at 2000 rpm for twenty minutes at room temperature. The mononuclear
cell layer was isolated, washed with phosphate buffered saline
(PBS) and centrifuged at 1400 rpm for ten minutes at 4.degree. C.
The cell pellet was incubated with a 1:10 dilution of
fluorescein-conjugated anti-TfR (anti CD 71) Becton-Dickinson
Catalog No. 7513) in PBS on ice for thirty minutes. The cells were
washed once in PBS prior to flow sorting.
[0133] Analysis and sorting were performed on a Becton-Dickinson
FACS IV with a Consort 40 program as described (Bianchi et al.,
Cytometry 8:197-202 (1987)). The gain was standardized manually
using fluorescent beads and a fluorescein isothiocyanate (FITC)
conjugated antibody control, keyhole limpet hemocyanin, an antigen
not expressed on human cells (Becton-Dickinson Catalog No. 9041). A
small aliquot of the woman's mononuclear cells were incubated with
the antibody control to determine background fluorescence.
TfR.sup.+ (fluorescent) and TfR.sup.- (non-fluorescent) cells were
determined by physical separation on a logarithmic scale and sorted
into 1.5 ml centrifuge tubes.
[0134] Fluorescence in Situ Hybridization
[0135] A solution of methanol and acetic acid (3:1) was used to fix
nuclei from sorted cells to glass slides which were then stored at
-20.degree. C. (Klinger et al., Am. J. Hum. Genet. 51:55-65
(1992)). The contents of the entire forementioned Klinger et al.
reference is expressly incorporated by reference. Prior to
hybridization, slides were warmed briefly at 60.degree. C. Control
hybridizations with male lymphocytes were performed
concurrently.
[0136] The probe sets used for hybridization to the Y chromosome
and to chromosome 21 have been described previously (Klinger et
al., cited supra, (1992)). The Y chromosome probe was labeled with
biotin-dUTP (Sigma) and the chromosome 21 probe was labeled with
digoxigenin-dUTP (Boehringer Mannheim). The probes were hybridized
simultaneously under suppression conditions (Cremer et al., Hum
Genet 80:235-246 (1988); Lichter et al., Hum Genet 80:224-234
(1988)). The Y chromosome probe was detected with avidin conjugated
to Texas Red (Vector Laboratories) and the chromosome 21 probe was
detected with anti-digoxigenin conjugated to fluorescein
isothiocyanate (FITC) (Boehringer Mannheim). The slides were
mounted in 2.33% DABCO (1,4-diazobicyclo[2.2.2]octane) (Sigma) in
100 mM Tris-HCl, pH 8.0, 90% (v/v) glycerol with 0.5 ug/ml
4,6-diamidino-2-phenyl-indole (DAPI) as a counterstain (Sigma).
Slides were analyzed using a Zeiss Axioplan epifluorescence
microscope. FITC and Texas Red were monitored simultaneously using
a dual band pass filter set (Omega Optical, Inc., Brattleboro,
Vt.). Images were captured with a cooled confocal digital camera
(Photometrics Ltd., Tucson, Ariz.) and image processing was
performed with software developed by Recognition Technology, Inc.,
(Westboro, Mass.).
[0137] The results of the contour plot depicting fluorescence
intensity versus light scatter are shown in FIG. 9. The percentage
of TfR.sup.+ cells was 1.3. A total of approximately 43,000
TfR.sup.+ cells and 300,000 TfR.sup.- cells were sorted.
[0138] In situ hybridization studies performed on the TfR.sup.-
cells revealed the presence of numerous nuclei. Control slides
prepared from male lymphocytes gave bright signals with both the 21
and Y probes. The nuclei from TfR.sup.- cells that hybridized to
the probes were exclusively maternal, with two signals for
chromosome 21 and no signal with the Y. The majority of the sorted
cells in the TfR.sup.+ fraction did not hybridize, due to the fact
that most of them were reticulocytes, which are anucleate. Most of
the nuclei present were in a few giant clumps that had retained
large amounts of cytoplasm. Of the few TfR.sup.+ cells that did
hybridize, FITC and Texas Red signals were generally weak. There
were three nuclei with one Texas Red and three FITC signals,
consistent with the presence of one Y chromosome and three copies
of chromosome 21 (FIG. 10). The remainder of the data are
summarized as follows.
2 Results of hybridization studies on scored cells: Texax Red (Y)
FITC (21) Number of Cells Number 1 3 3 of signals 1 0 8 0 3 1 1 1 1
13 Uninformative 15 28
[0139] Equivalents
[0140] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *