AU770015B2 - Separation of foetal trophoblasts from maternal blood - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

S..

Name of Applicant: Actual Inventor: Address for Service: Invention Title: Phoenix Medical Limited Patricia Mary Beckett Robertson CULLEN CO., Patent Trade Mark Attorneys, 239 George Street, Brisbane, QId. 4000, Australia.

Separation of Foetal Trophoblasts from Maternal Blood The following statement is a full description of this invention, including the best method of performing it known to us: This is a further application made under Section 79B of the Patents Act 1990 for a patent for an invention disclosed in our co-pending Australian patent application no. 733273 entitled "Body Fluid Testing". The disclosure of that application is incorporated herein by reference.

This invention is concerned with the separation of blood into various of its components, to enable a variety of tests to be performed. In particular, the invention is directed to the use of a filter to separate foetal trophoblasts from maternal blood.

In the field of haematology and related areas such as gene therapy and forensic pathology, and in other fields, such as the determination of sperm fertilising potential and cell separation from bone marrow, hitherto it has involved complex and expensive apparatus and procedures, and has required considerable operator skills to separate correctly the components of the particular body fluid. This has been particularly so with blood, consequent i 15 on the need for careful preparation of a blood sample.

In particular, when testing or screening of a foetus for one or more of a number of possible abnormalities, it has hitherto predominantly been effected by invasive techniques frequently to the discomfort of the mother.

20 It is the object of the invention to provide a method of separating foetal trophoblasts from maternal blood using apparatus of notable simplicity and relatively low cost, with attendant procedures well within the capabilities 0: of junior members of laboratory staff.

This invention involves the use of a filter for separating foetal 0 25 trophoblasts from maternal blood.

In one form, the invention provides a method for separating foetal trophoblasts from maternal blood wherein a sample of maternal blood is applied to a filter. The filter permits the passage of maternal blood cells, but entraps foetal trophoblasts.

Typically, a sample of maternal blood is applied to one side of the filter, and a vacuum is applied to the opposite side of the filter to draw the maternal blood through the filter and leave the foetal trophoblasts trapped on the filter. The filter is suitably pre-treated, e.g. gel coated, to prevent the adherence of the entrapped component to the filter. The trapped foetal cells may be released from the filter in situ or following removal of the filter from its associated separation apparatus. Alternatively, tests may be effected on the cells while they remain on the filter.

Co-pending Australian patent application 733273 discloses a method of separating the DNA content of blood, in which a whole blood sample is applied to one side of a filter, vacuum is applied to the opposite side of the filter to draw the whole blood sample therethrough and separate the plasma and red cell content of the blood from the leukocyte cell content, leaving the leukocyte content trapped in the filter, followed by the application of water or, preferably, isotonic saline, to the same side of the filter to be drawn therethrough by the vacuum applied to the opposite side of the filter to lyse the leukocyte cells trapped in the filter. As an alternative to. the 15 application of water/isotonic saline, a chemical lysing agent or compatible cell detergent can be used to separate the plasma and red cell content of the blood from the leukocyte cell content, and to leave the leukocyte content trapped in the filter. Following lysing a removable end cap on the chamber now containing what is waste material can be removed and discarded, the 20 chamber inverted and isotonic saline applied to the opposite side of the filter to wash out the cell contents of the leukocyte cells from the filter into an appropriate receptacle from where the DNA content is removed. The suitable receptacle may be the removable end cap on the other end of the chamber.

Alternatively, following removal of the end cap containing waste material, it can be replaced by the end cap from the opposite side of the chamber, and isotonic saline can be applied to the same side of the filter to wash out the cell contents of the leukocyte cells into the clean end cap, from where the DNA content can be removed. As a still further alternative, it is possible for certain tests to leave the leukocyte content trapped in the filter and to serve as a test base for a variety of medical tests.

In the circumstance where it is the removal of leukocyte cells, it is preferred that the commercially available filter known in trade as Pall LK4 is used. It is also preferred that the end cap which receives the cell contents is provided with a suitable membrane to isolate the DNA content of the cells from other cell debris washed from the filter to provide a clean DNA sample able to be liberated from the membrane utilising conventional transfer techniques. The separation method, in its application to DNA harvesting and leukocyte testing, by its equipment and its method avoids completely the need for any preparation of a blood sample, and allows the separation, collection, and gathering of the DNA content in exceedingly simple and efficient manner.

Whole blood may simply be applied to the filter through the open end of the chamber, without pre-preparation, and vacuum applied to the opposite side such as by a relatively simple laboratory vacuum pump, or by, for example a standard medical syringe. This causes red cells and plasma to be drawn through the filter and collected in a removable end cap, leaving 15 leukocytes trapped in the filter. With continued application of vacuum, water, isotonic saline, or a chemical lysing agent, or a compatible cell detergent is then applied to and drawn through the filter, causing chemical lysing, or lysing of the leukocytes by osmotic shock.

Once the cell contents have been collected, the DNA 20 component can then be gathered by conventional techniques.

In other spheres of testing such as, for example, foetal testing, sperm counting, bone marrow testing, urine, and saliva testing, it is desirable to provide multiple samples. Here, a sample is applied to each of the number of filters overlying a respective waste collection chamber on the second plate 25 following which the first plate is applied to the second plate to trap the filters and the assembly of first and second plates applied to the third vacuum forming plate.

This invention of this application is concerned particularly with the screening and isolation of foetal trophoblasts (immature blood cells) from maternal blood by using a filter that allows the passage of maternal blood cells and which entraps foetal blood cells. For example, a Pall J100 or similar membrane, with the gel-coated side positioned to receive the blood sample, may be used. The filter is preferably coated to render it non-stick, as is the sample collection chamber. That is, the filter is pre-treated to prevent the adherence to the filter of the entrapped component.

A whole blood sample taken from a mother need not be the subject of any prior preparation and can be applied to each of the number of filters. With vacuum applied via the vacuum chamber to the waste collection chamber, maternal blood cells of the blood are drawn through the filter and collected in the waste collection chamber, whilst foetal blood cells are left entrapped in the filter. The second and third plates are removed and the waste material in the waste collection chambers discarded, and the first plate inverted. Here again, it is preferred that the foetal cells are released from the filter into the collection chambers from where they can be removed for test. It is, however, possible that the foetal filters can be stripped of debris to leave the cells trapped in the filter and tests effected on the cells whilst on the filter.

S 15 In its preferred form, a procedure for isolating and screening foetal trophoblasts comprises isolating foetal blood cells from a maternal blood sample using a specially prepared membrane which allows the S. maternal blood cells to pass through the membrane leaving the foetal blood cells trapped within the filter, the foetal blood cells then being lysed and a S* 20 chemical and specific antibody marker compatible with specific chromosomes added, the marker having the ability to fluoresce under shortwave ultra violet light to signal an abnormality in the foetal cells. Thus, as in the example of Downs Syndrome, chromosome 21 will be shown to be present in triplicate, instead of in duplicate. The chromosome 21 will appear on the screen under 25 UV light as three fluorescent dots.

is envisaged that this non-invasive screen could be carried out from 10 weeks gestation. The technique can be used for any other genetically carried syndromes i.e. Huntingdons chorea, cystic fibrosis and spina bifida.

A preferred embodiment of the invention will now be further described, by way of example only, with reference to the accompanying drawings, in which:- Figure 1 is a perspective view of a first embodiment of 6 apparatus suitable for body fluid separation; Figure 2 is an exploded perspective view of a second embodiment of apparatus for multi-sample body fluid testing; and Figure 3 is a section on the line 111-111 of Figure 2, in its assembled condition.

In Figure 1 a body fluid separation means particularly suited to DNA harvesting is formed by a chamber 1 having upper and lower sectors 2, 3, and a centrally disposed filter 4. Each of the upper and lower sectors 2, 3, is provided with a removable end cap 5, 6, respectively, each end cap being in sealing engagement with the respective end of the chamber sector. Each sector has a connection 7, 8 closed by a respective plug 9, 10, to enable either or both of the chamber sectors 2, 3, to be connected to a source of vacuum.

At the outset, the end cap 5 is removed and a whole blood 15 sample introduced into the chamber and on to the filter 4, such as by way of a conventional syringe. The end cap 5 is replaced, the plug 10 removed from the connection 8 on the lower sector 3 of the chamber, and the connection 8 connected to a source of vacuum, which again may be by way of a conventional syringe, the presence of vacuum below the filter causing blood S: 20 to be drawn through the filter. The filter is specifically chosen to enable the leukocyte content of the blood to be trapped on it, the plasma and red cell content of the blood drawn through the filter being deposited in the end cap 6.

Following this, the end cap 5 is removed and water, isotonic saline, an appropriate chemical lysing agent or a compatible cell detergent is applied to S 25 the filter and drawn through the filter by the vacuum existing below it and the end cap 5 then replaced. The cap 6 is removed and the waste material contained in it discarded, following which the container can be inverted, the plug 10 is replaced on the connection 8, the plug 9 removed and the connection 7 connected to vacuum. With water or isotonic saline then applied to the opposite side of the filter, the cell contents of the leukocyte cells are washed out to be gathered in the cap 5. Following this the cap 5 can be removed to allow the harvesting of DNA content. As an alternative to this, and following the removal of the end cap 6, the end cap 5 can be applied to the container in place of the end cap 6, water, isotonic saline, a chemical lysing agent, or compatible cell detergent applied to the same side of the filter to wash out the cell content of the leukocyte cells into the end cap 5 by the continued application of vacuum through the connection 8.

Whichever of the caps 5, 6, is employed to gather the cell contents of the leukocyte cells, it is most desirable that a filter membrane 11 is provided to isolate the DNA content of the cells from any other cell debris washed from the filter, the membrane being readily removable from the end cap for the subsequent stripping of the isolated DNA content.

In Figures 2 and 3 body fluid separation means for the simultaneous treatment of a number of samples is shown. Although the embodiment illustrates the treatment of six samples simultaneously, it will be appreciated that any number of samples can be dealt with. In this 15 embodiment, there is provided a first plate 12 formed with six collection chambers 13, and a second plate 14 formed with six cooperating waste receiving chambers 15. Each waste receiving chamber 15 has a connection port 16 to connect each waste receiving chamber to a vacuum chamber 17 formed in a third plate 18, the third plate 18 having a connection 19 to a source of vacuum (not shown). Contained in the vacuum chamber 17 is a membrane 20. Thus with the second plate 14 located on the third plate 18, and with individual filters 21 positioned over each waste receiving chamber, a sample of body fluid is applied to each filter, and the first plate 12 positioned on the second plate to provide the assembly as is indicated by Figure 3. With 25 vacuum applied through 19 to the chamber 17 a vacuum is applied through each port 16 to the waste collection chamber, to cause the plasma/fluid content of the sample on the filter to be drawn through the filter into the waste receiving chamber 15, from where it passes through the port 16 for collection on the membrane 20. The third plate 18 is removed and the waste material on the membrane 20 discarded, the first and second plates inverted and the second plate removed to allow access to the filters and to enable the cells or other body fluid components trapped on the filter to be washed into the sample collection chambers 13.

Such an apparatus as is illustrated by Figures 2 and 3 is eminently suited to, such as for example, the screening and isolation of foetal trophoblasts from maternal blood, an appropriate membrane/filter being employed that is gel coated, the blood sample being applied to the gel-coated side, and the membrane/filter together with each collection chamber being further treated to render it non-stick, it being known that foetal trophoblasts readily adhere to other substances. Once the foetal trophoblasts have been isolated, a genetic probe can be applied to the cells which probe is specific to a given chromosome, for example chromosome 21 which, if it appears in triplicate instead of duplicate, confirms a diagnosis of Down's Syndrome. The technique of fluorescent in situ hybridisation, either interphase or metaphase, probes can be used to identify all known genetic disorders. In situ fluorescence can be visualised with a simple microscope or ultra-violet i 15 reader, when the probe specific to the chromosome to be examined would be seen as fluorescent dots two dots Down's negative and three dots Down's positive).

Claims (9)

1. A method of separating foetal trophoblasts from maternal blood comprising using a filter and applying a sample of maternal blood to the filter, wherein the step of using a filter comprises using a filter which is configured to permit the passage of maternal blood cells and the entrapment of foetal trophoblasts.

2. The method of claim 1, wherein maternal blood cells of the blood are drawn through the filter.

3. The method of claim 2, wherein the maternal blood cells of the blood are drawn through the filter by the application of vacuum to the filter.

4. The method of claim 3, wherein the sample of maternal blood is applied to one side of the filter and vacuum is applied to the opposite side of the filter to draw the maternal blood through the filter and leave the foetal trophoblasts trapped on the filter.

The method of claim 4, wherein the foetal trophoblasts are released from the filter.

6. The method of claim 4, wherein a body fluid separation means is used, comprising a first plate formed with two or more collection chambers, a second plate having a corresponding number of waste receiving chambers, a connection to vacuum for each of said waste receiving chambers, a filter between each collection chamber °20 and cooperating waste receiving chamber, and a third plate forming a vacuum chamber to connect each waste receiving chamber to vacuum.

7. The method of any one of claims 1 to 6, wherein the filter is pre-treated to prevent the adherence to the filter of the entrapped component.

8. The method of claim 7, wherein the filter is gel coated. 5

9. A method of separating foetal trophoblasts from maternal blood, substantially as hereinbefore described. oooole Apparatus substantially as hereinbefore described when used for separating foetal trophoblasts from maternal blood. DATED this twelfth day of August 2003 Phoenix Medical Limited By their Patent Attorneys Cullen Co. r