Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-08
2001-12-18
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S002000, C435S004000, C435S005000, C435S007800, C435S007210, C435S320100, C435S194000, C435S196000, C435S007100, C435S252300, C435S242000, C435S975000, C435S810000, C435S091200, C435S007240, C435S007250, C435S040500, C435S007500, C435S007720, C435S007900, C435S007920, C436S063000, C436S519000, C436S520000, C436S524000, C436S510000, C436S518000, C436S527000, C436S066000, C436S080000
Reexamination Certificate
active
06331395
ABSTRACT:
The present invention relates to diagnostic methods, in particular to methods of prenatal diagnosis and to reagents for use in such methods.
Prenatal diagnosis is carried out widely in hospitals throughout the world. Existing procedures such as fetal, hepatic or chorionic biopsy for diagnosis of chromosomal disorders including Down's syndrome, as well as single gene defects including cystic fibrosis are very invasive and carry a not inconsiderable risk to the foetus and a small risk to the mother.
Amniocentesis, for example, involves a needle being inserted into the womb to collect cells from the embryonic tissue or fluid. The test, which can detect Down's syndrome, carries a miscarriage risk estimated at 1%.
Fetal therapy is in its very early stages and the possibility of very early tests for a wide range of disorders would undoubtedly greatly increase the pace of research in this area. Current fetal surgical techniques have improved, making fetal surgery for some genetic problems like spina bifida and cleft palate very feasible. In addition, relatively simple effective fetal treatment is currently available for other disorders for example 21-hydroxylase (treatment with dexamethasorie) and holocarboxylase synthetase (treatment with biotin) deficiencies, as long as detection can take place early enough.
Thus, relatively non-invasive methods of prenatal diagnosis are an attractive alternative to the very invasive existing procedures. A method based on maternal venepuncture should make earlier diagnosis more widely available in the first trimester, increasing options to parents and obstetricians (because genetic disorders could be detected earlier and more safely), and allowing the eventual development of specific fetal therapy.
The possibility of recovering fetal cells from the maternal circulation has excited general interest as a possible means, non-invasive to the fetus, of diagnosing fetal anomalies (Simpson & Elias (1993)
J. Am. Med. Assoc.
270, 2357-2361). Initial interest was directed towards trophoblastic detection systems but separation of those cells by flow cytometry has been unreliable as maternal lymphocytes appeal to absorb proteins released by trophoblastic cells (Mueller et al (1990) Lancet 336, 197-200; Covone et al (1984) Lancet 13 October edition, 841-843). More recently, attention has focused on the development of methods to isolate fetal blood cells for cytogenetic analysis particularly nucleated fetal erythrocytes as their numbers exceed those of fetal lymphocytes in the maternal circulation. Identification of fetal red blood cells in maternal blood has been described ie in a male fetus with Y centromere probes to identify fetal cells or amplification of Y-specific DNA sequences (Price et al (1991)
Am. J. Obstet. Gynecol.
165, 1731-1735; Zheng et al (1993)
J. Med. Genet.
30, 1051-1056; Hamada et al (1993)
Hum. Genet.
91, 427-432; Cheung et al (1996)
Nature Genetics
14, 264-268; and Williamson (1996)
Nature Genetics
14, 239-249) or karyotype identification in trisomic conditions (for example, see Bianchi et al (1992)
Hum. Genet.
90, 368-370).
Hume et al (1995)
Early Human Development
42, 85-95 shows that the microsomal glucose-6-phosphatase enzyme protein is present in human embryonic and fetal red blood cells.
Pazouki et al (1996)
Acta histochem
. (Jena) 98, 29-37 attempts to identify fetal nucleated red blood cells using combined immunocytochemistry using a human fetal haemoglobin antibody and an in situ hybridisation method using X and Y chromosome probes.
Hume et al (1996)
Blood
87, 762-770 describes study of the expression of endoplasmic reticulum proteins in human embryonic and fetal red blood precursors.
Wachtel et al (1991)
Human Reproduction
6, 1466-1469 describes the use of PCR to identify Y-specific DNA sequences in maternal cells isolated by cell sorting with transferrin receptor antibody and glycophorin A antibody.
Yeoh et al (1991)
Prenatal Diagnosis
11, 117-123 describes the detection of fetal cells in the maternal circulation by enzymatic amplification of a single copy gene that was fetal specific.
Holzgreve et al (1992)
J. Reprod. Med.
37, 410-418 shows that the transferrin receptor antigen alone is not sufficient for enrichmnent of fetal nucleated erythrocytes and points out that the reproducibility and reliability of the techniques are still limited, mainly due to the lack of very specific cell markers.
Zheng et al (1993)
J. Med. Genet.
30, 1051-1056 describes the use of a magnetic activated cell sorter (MACS) to enrich fetal nucleated erythrocytes using mouse monoclonal antibodies specific for CD45 and CD32 to deplete leucocytes from maternal blood. The paper points out that significant maternal contamination was present even after MACS enrichment preventing the accurate analysis of fetal cells by interphase fluorescence in situ hybridisation (FISH).
Tomoda (1964)
Nature
202, 910-911 describes the demonstration of fetal erythrocytes by immunofluorescent staining.
There exists a need for improved methods for identifying fetal cells in maternal blood in order to carry out prenatal diagnosis.
We attempted to isolate embryonic and fetal nucleated red blood cell from maternal blood with the established immuno-magnetic sorting using sequentially anti-CD45 and anti-CD18 antibodies to remove white blood cells and then anti-CD71 (transferrin receptor) antibodies to enrich fetal nucleated red blood cells. We were very disappointed to discover that immuno-magnetic sorting, with the anti-CD71 antibody, did not purify embryonic red blood cells of the megaloblastic series. The advantage of purifying megaloblastic cells is that they are the predominant red blood cell type in the embryo and early fetus and that they are nucleated whereas the vast majority of adult red blood cells are normocytic and non-nucleated.
In subsequent conventional immunohistochemistry, we found that CD71 interactions with megaloblasts were very weak presumably explaining the poor purification of embryonic cells with this antibody. This is a very major problem in the current use of maternal blood for early diagnosis. We have shown that the nucleated megaloblastic series predominates in early development compared to nucleated normoblasts. This means that using the conventional antibodies ie anti-CD71, it is very difficult to obtain pure nucleated haemopoeitic cells arising from the early conceptus. Anti-CD71 does not immunoreact with the majority of these early cells making this technique possible in specialised research labs with specialist staff and equipment, but not practical in routine service laboratories.
The use of anti-CD71 is described in Cheung et al (1996)
Nature Genetics
14 264-268 and is reviewed by Williamson (1996)
Nature Genetics
14, 239-240. Having to look at thousands of cells to find a few fetal ones on slides is a problem with this approach, as is the fact that anti-CD71 antibodies are not selective enough for fetal cells and do not react with embryonic cells.
An object of the present invention is the provision of improved methods for identifying fetal cells in, and isolating them from, maternal blood. In particular, an object of the invention is the provision of methods of identifying and isolating embryonic or early fetal red blood cells, and analysing the cells for fetal abnormalities.
A first aspect of the invention provides a method of identifying embryonic or fetal red blood cells in a sample containing natural blood cells and embryonic or fetal red blood cells or both, the method comprising determining which cell or cells contain or express an adult liver component.
A second aspect of the invention provides a method of isolating embryonic or fetal red blood cells from a sample containing maternal blood cells and embryonic or fetal red blood cells or both, the method comprising isolating the cells which contain or express an adult liver component.
It has not previously been proposed that embryonic or early fetal nucleated red blood cells are functioning as adult liver cells while circulating in the bloodstream
Burchell Ann
Hume Robert
Chin Christopher L.
Cook Lisa V.
Farber Michael B.
Oppenheimer Wolff & Donnelly LLP
The University of Dundee
LandOfFree
Prenatal diagnostic methods does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Prenatal diagnostic methods, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Prenatal diagnostic methods will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2573744