Claims: , Description:SUMMARY OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Generally, nomenclatures utilized in connection with, and techniques of, cell and molecular biology and chemistry are those well-known and commonly used in the art. Certain experimental techniques, not specifically defined, are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.
For purposes of the clarity, following terms are defined. The term “prenatal diagnosis” as used herein includes determination of any disease or abnormality in a foetus by means of foetal DNA analysis from maternal serum or plasma. Included are, detection of foetal abnormalities such as chromosomal aneuploidies or simple mutations, sex linked genetic defects as well as sex determination. The term “non-invasive “as used herein refers to the use of techniques that do not physically invade the space occupied by the foetus or related tissue such as the placenta. Additionally, it refers to screening of maternal blood sample obtained from a pregnant female during early stages of pregnancy.
A chromosomal abnormality can be numerical or structural, and includes but is not limited, to aneuploidy, polyploidy, inversion, a trisomy, a monosomy, duplication, deletion, deletion of a part of a chromosome, addition, addition of a part of chromosome, insertion, a fragment of a chromosome, a region of a chromosome, chromosomal rearrangement, and translocation. Chromosomal abnormality may also refer to a state of chromosomal abnormality where a portion of one or more chromosomes is not an exact multiple of the usual haploid number due to, for example, chromosome translocation. Chromosomal translocation (e.g. translocation between chromosome 21 and 14 where some of the 14th chromosome is replaced by extra 21st chromosome) may cause partial trisomy 21. A chromosomal abnormality can be correlated with presence of a pathological condition or with a predisposition to develop a pathological condition. A chromosomal abnormality may be detected by quantitative analysis of nucleic acid. Preferably, the term “chromosomal aneuploidy or abnormality ” used herein refers to trisomy conditions like Down syndrome and Edwards’ syndrome or any other genetically inherited autosomal or sex linked disorders. “Enrichment” is defined herein as increasing the relative concentration of foetal cells to maternal cells in a sample, preferably while maintaining a high yield of foetal cells. Preferably, cells are enriched by removal of 70%, 80%, 90%, or 99%, preferably, 99% of non-foetal cells.

As illustrated in Figure 1 the following embodiment discloses a method for detecting chromosomal abnormality, e.g. chromosomal aneuploidy, of a foetus, consisting essentially of the steps:
1. Taking maternal blood sample, preferably, within 15-20 weeks of pregnancy;
2. Enriching foetal cells from maternal blood by Magnetic activated cell sorting (MACS)
3. Fixing the enriched cells to a slide
4. Subjecting the cells on the slide to KB staining
5. Analyzing the cells under a microscope and marking foetal cells.
6. Evaluating the marked foetal cells by Fluorescence In Situ Hybridization (FISH) for detection of chromosomal abnormalities OR
7. Picking individual marked foetal cells for DNA extraction for detection of chromosomal abnormalities by Next Generation Sequencing (NGS).
In a broad aspect, the present disclosure relates to methods for detection of any defects in/ or monitoring the health of the foetus or determination of sex of the foetus by testing maternal blood taken between 15-20 weeks of pregnancy. Preferably, maternal blood is tested by the methods disclosed herein, within fifteen to twenty weeks of pregnancy. In the present text, methods for detection of any chromosomal changes which may confer a disease phenotype to the foetus are described. Further, the present subject matter relates to methods for determination of foetal-derived paternally-inherited polymorphisms or mutations or genes as in the case of sex linked disorders. Alternatively, the methods described herein may be further employed for detection of autosomal dominant as well as recessive genetic disorders.

Examples of foetal abnormalities that can be diagnosed by the methods of the present invention include, but are not limited to, trisomy 13, trisomy 18, trisomy 21 (Down syndrome), Klinefelter Syndrome (XXY) and other irregular number of sex or autosomal chromosomes. Furthermore, the methods may be used to distinguish maternal trisomy from paternal trisomy, and total aneuploidy from segmental aneuploidy. Additionally, the methods may be used to identify monoploidy, triploidy, tetraploidy, pentaploidy and other higher multiples of the normal haploid state. In some embodiments, the maternal or paternal origin of the foetal abnormality is determined.

In an embodiment, a method for detection of genetic disorders in foetus includes making use of foetal cells circulating in mother’s blood. In a preferred embodiment, the method utilizes foetal-nucleated RBCs (fnRBCs) for detection of genetic disorders. In another embodiment, the present method enriches foetal cells from maternal blood by positive selection making use of foetal cell specific biomarkers. In yet another embodiment, the present method enriches foetal cells from maternal blood by positive and negative selection making use of foetal cell specific biomarkers.

In a preferred embodiment, the foetal specific biomarkers include surface antigens that are expressed on surface of foetal nucleated erythroid cells .The foetal specific biomarkers chosen for enrichment of foetal cells include but are not limited to glycophorin A (CD 235a) , transferrin receptor, CD71, CD 36, and the like. In an aspect, the foetal specific biomarkers chosen for positive selection are CD 71 and CD 235a. In a preferred aspect, foetal nucleated red blood cells are enriched by means of positive selection for CD71 and CD 235a antigen using Magnetic Activated Cell Sorting (MACS).

In an embodiment, once the foetal cells have been enriched, they are fixed to a surface such as a slide. Further, the cells are fixed to a slide with the help of a fixative. In another embodiment the fixative used is ethanol or mannitol or glycerol or any other precipitating fixative. In a preferred embodiment, the fixative used is 80% ethanol. Following this fixation, the cells of foetal origin are characterised and marked based on difference between foetal RBCs and maternal RBCs. Haemoglobin from adult erythrocytes gets eluted in acidic medium whereas foetal RBC is resistant to it. This forms the basis of a staining method termed as Kleihauer- Betke (KB) staining which is conventionally used for detection of foetal maternal haemorrhage.

In an embodiment, the present method utilizes KB staining method for distinguishing foetal and maternal RBCs. Incubation of cells in acidic buffer such as citric phosphate buffer solution followed by washing leads to elution of haemoglobin from maternal adult RBCs while foetal RBCs retain foetal haemoglobin. The slide is then subject to staining by haematoxylin and erythrosine B sequentially which stains haemoglobin within foetal RBCs which appear as pink spots while adult RBCs lacking haemoglobin do not take up the stain. This helps in identification of foetal cells which appear rose pink in colour from adult RBCs appear as “ghosts”. Following this, the foetal cells can be marked on the slide. In an embodiment, the characterized and marked foetal nucleated RBCs (fnRBCs) may then be subjected to foetal DNA analysis for detection of various probable genetic defects by any of the several methods known to person skilled in the art. In particular, it is envisaged that methods for detection of genetic defects include but are not limited to PCR, hybridization methods, Next Generation Sequencing and SNP detection. In a preferred embodiment, stained and marked foetal cells can be picked individually and subject to foetal DNA analysis following DNA extraction. By doing so, we can hereby, accept genome analysis results with greater confidence since the background caused due to mother’s cells has been eliminated. The methods, compositions, and kits provided herein provide diagnostically relevant information as well as a basis for detection of genetic disorders in foetus during early stages of pregnancy. In an embodiment, the assays provided herein, determine whether a foetus has any genetic disorder. In another embodiment, the assays provided herein, characterize the genetic disorder, if any, in a foetus.

The present assay is further illustrated by the following examples which, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realising the assay in diverse forms thereof.

Example 1
Obtaining maternal blood sample preparation
Maternal whole blood was collected in EDTA vacutainer and diluted three times with dilution buffer (1x PBS, 2.5 mM EDTA). 7 ml of diluted blood was carefully layered on 3 ml of Histopaque solution in a 15ml centrifuge tube and centrifuged at 400g for 20 min at room temperature (RT).The plasma layer was then carefully removed and discarded. The Buffy coat (Peripheral Blood Mononuclear Cells- PBMCs) was collected in new 15 ml centrifuge tube and washed with 10ml wash buffer (1x PBS, 2.5 mM EDTA, 5% FBS) . This was followed by another round of centrifugation at 200g for 10 min at RT. The supernatant was discarded and the washing step was repeated. Finally, the cell pellet was re-suspended in 1 ml MACS buffer (1x PBS, 2.5 mM EDTA, 5% FBS).Cell count was determined by mixing the 10µl of cell suspension with 10µl Trypan blue dye and viewing cells on a Haemocytometer / Neubauer’s Chamber.

Example 2
Foetal cell enrichment
The cell suspension obtained in Example 1 was centrifuged at 300g for 10 minutes at RT. The supernatant was discarded completely and the cell pellet was re-suspended in 80µl of MACS buffer per 107 cells. This was followed by addition of 20µl of CD71 Microbeads per 107 cells. After mixing well, the mixture was incubated at 40-80C for 15 minutes in dark. Thereafter, washing was done by adding 2 ml MACS buffer per 107 cells and centrifuging at 300g for 10 minutes at RT. The supernatant was discarded and the cell pellet was re-suspended in 500µl of MACS buffer per 108 Cells. Column loaded with 500 µl re-suspended cell suspension was placed in the magnetic field of a suitable MACS separator and 15ml tubes was kept below the column to collect the eluent. Prior to loading the cell suspension, the column was rinsed with appropriate amount of MACS buffer (MS: 500µl, LS: 3ml) Table1 depicts the details of sample loading in different columns.

Table1.
Column Max. number of labelled cells Max. number of total cells
MS 107 2 X 108
LS 108 2 X 109
XS 109 2 X 1010

The unlabelled cells (CD71-) that passed through the column were collected in 15 ml tubes lying underneath, and discarded. Following this, the column was washed with appropriate quantity of wash buffer (MS: 3 X 500µl, LS: 3 x 3ml).The column was removed from the separator and placed on a new 15ml tube. An appropriate amount of buffer (MS: 1ml, LS: 5ml) was added onto the column and the fraction with magnetic labeled cells was immediately flushed out by firmly applying the plunger supplied with the column. This was the CD71+ cell fraction. Cell count was determined by mixing the 10µl of cell suspension with 10µl Trypan blue dye and viewing cells on a Haemocytometer / Neubauer’s Chamber. Then, cell suspension was centrifuged at 300g for 10 min at RT. Supernatant was discarded and the cell pellet was re-suspended in 80µl of MACS buffer per 107 cells. Following this, 20µl of CD235a microbeads were added per 107 cells, mixed well and incubated at 40-80C for 15 minutes in dark. Washing step was repeated by adding 2 ml MACS buffer per 107 cells and centrifuging at 300g for 10 minutes at RT. The supernatant was again discarded and the cell pellet was re-suspended in 500µl of MACS buffer per 108 cells. Column loaded with 500 µl re-suspended cell suspension was placed in the magnetic field of a suitable MACS separator and 15ml tubes was kept below the column to collect the eluent. Prior to loading the cell suspension, the column was rinsed with appropriate amount of MACS buffer (MS: 500µl, LS: 3ml) Table 2 depicts the details of sample loading in different columns.

Table 2.
Column Max. number of labelled cells Max. number of total cells
MS 107 2 X 108
LS 108 2 X 109
XS 109 2 X 1010

The unlabelled cells (CD71+ CD235a-) that passed through the column were collected in 15 ml tubes lying underneath, and discarded. Following this, the column was washed with appropriate quantity of wash buffer (MS: 3 X 500µl, LS: 3 x 3ml). The column was removed from the separator and placed on a new 15ml tube. An appropriate amount of buffer (MS: 1ml, LS: 5ml) was added onto the column and the fraction with magnetic labeled cells was immediately flushed out by firmly applying the plunger supplied with the column. This was the CD71+ CD235a+ cell fraction. Cell count was determined by mixing the 10µl of cell suspension with 10µl Trypan blue dye and using Hemocytometer / Neubauer’s Chamber and this cell fraction was stored for further steps.

Example 3
Identification and marking of foetal cells by Kleihauer-Betke staining
Citrate Phosphate Buffer was pre-warmed at 370C in a Coplin jar or staining dish. Using clean, labelled microscope slides, thin blood smears were prepared. Control slides were prepared using positive HbF blood (cord-blood) and normal adult blood while prepare TEST slides were prepared using the enriched (CD71+ CD235a+ ) cells. Following this, slides were air-dried for approximately 10 minutes and fixed by immersing in Ethanol Fixative (80% Ethanol) for 5 minutes. Thereafter, slides were rinsed thoroughly with tap water and air-dried. Then, TEST and CONTROL slides were immersed in pre-warmed Citrate Phosphate Buffer Solution at 370C for 5 minutes and agitated after 1 and 3 minutes of immersion. Following this, they were rinsed thoroughly with distilled water and air dried completely. The slides were stained in Acid Hematoxylin Solution at RT for 3 minutes followed by rinsing with distilled water and shaking off excess water. Slides were further counterstained in 0.1% Eosin B Solution at RT for 4 minutes followed by rinsing with distilled water and air drying. Dry coverslip was placed on the slide and examined using oil immersion (1000X).

Example 4
Detection of chromosomal abnormality by Fluorescence In Situ Hybridization
The enriched cell pellet of Example 2 was re-suspended in 20ul MACS buffer and incubated with 5ml of 0.56% KCL Solution at RT for 30 minutes. After incubation, the cell suspension was centrifuged at 226 g for 10mins at RT following which, the supernatant was discarded. Thereafter, 5 ml of freshly prepared chilled Fixative (1:1) was added drop wise to the centrifuge tube containing the cell pellet and again centrifuged at 226 g for 10mins at RT. The supernatant was discarded and the previous step was repeated. Following this, cells were fixed on a clean and dry frosted glass slide and observed under a phase contrast microscope. Well separated cells as seen under the microscope were marked with a diamond pencil. Slides were then exposed to working pepsin solution for approximately 20 seconds and washed in 2X Standard Sodium Citrate (17.532% NaCl, 8.823% Na3C6H5O7 ) solution for two minutes at RT. Washing step was repeated and slides were immersed slides consecutively in 70%, 85%, 100% ethanol coupling jars for 2 minutes each at RT. Probe solutions were prepared by centrifuging at 226 g for 2 minutes at RT, and tapping the solution for proper mixing .This process was repeated three times. Here onwards, all the subsequent steps were performed in dark.1µl of each probe was applied to the target area taking care that no air bubbles enter, followed by placing cover slips and sealing them with rubber cement. The slides were denatured at 750C on Hot Plate for 5 minutes followed by incubation in a Hybridization Chamber that had been pre-warmed at 370C for 16-18 hrs to allow the probe to hybridize to its target. Subsequently, freshly prepared wash solution1 (1ml 20XSSC, 0.15ml Igepal, 49ml D/w) was prewarmed by incubating in a water bath set at 750C for 1-2 hr 30-60 minutes while wash solution 2 (5ml 20XSSC, 0.05ml Igepal, 45ml D/w ) was kept at room temperature. Thereafter, rubber cement was removed from the slides and they were washed in Wash solution 2 for 30-40 seconds at RT followed by washing in wash solution 1 at 750C for 1 min 40 seconds and again leaving in wash solution 2 for 20 seconds at RT. Finally slides were removed from was solution 2 and edges were wiped. Then 8µl of DAPI counter stain was applied on a cover slip, onto which, the slide was inverted such that the hybridization area of the slide was covered with the cover slip. Slide was then examined under a fluorescence microscope with a proper filter sets.