WO1996007913A1

WO1996007913A1 - Diagnostic apparatus

Info

Publication number: WO1996007913A1
Application number: PCT/GB1995/002118
Authority: WO
Inventors: David Miller
Original assignee: University Of Leeds
Priority date: 1994-09-06
Filing date: 1995-09-06
Publication date: 1996-03-14
Also published as: AU3397095A; GB9417912D0

Abstract

Diagnostic apparatus comprises a light transmitting slide having a cell type conjugate immobilised on the surface; means for applying a suspension of cells to the surface, the substrate being adapted to bind cells contained in the suspension having a predetermined characteristic. The substrate may be etched, sintered or may carry immobilised beads to enhance the density of the bound cells.

Description

DIAGNOSTIC APPARATUS

This invention relates to diagnostic apparatus in use of which living cells of a particular type are isolated for detection by microscopy.

The cells which the apparatus may be adapted to isolate include specific sub-groups of leucocytes, populations of cells in disaggregated tissues, malignant cells from peripheral circulation or disaggregated tissues or fetal cells from the maternal circulation. The invention may be illustrated with reference to detection of fetal abnormalities.

A number of serious genetic or developmental illnesses can be detected while a growing fetus is only a few weeks old. Physical abnormalities can be detected by ultrasound screening but this is only effective for certain types of defects and requires expensive equipment and highly skilled personnel. Amniocentesis is an alternative wherein a sample of amniotic fluid surrounding the growing fetus is removed and the cells from the fluid are analysed. This is an accurate procedure, but requires skilled personnel, is expensive and does not provide immediate results. More significantly it is unpleasant for the mother and causes spontaneous abortion in a small proportion of tests.

Biochemical tests on a mother's blood are used for detection of spina bifida and Downs syndrome. Unfortunately current techniques do not allow detection of defects before the thirteenth week of pregnancy, at which stage development has advanced and decisions regarding termination of an affected fetus are more difficult. In addition the present tests are not perfectly diagnostic but merely indicate a raised likelihood of abnormality. Ultrasound scanning, amniocentesis or chorionic villus sampling must be undertaken as a further check.

Most genetic defects cannot be detected by current biochemical methods, however as early as the fifth week of pregnancy the blood of pregnant women contains cells derived from the fetus. Some of these fetal cells are nucleated red cells and trophoblasts which are normally absent in the maternal blood stream. These nucleated fetal cells can be analysed for fetal defects but they occur in maternal blood at an extremely low frequency, for example one in ten million. Various methods are therefore in development aimed at separating or enriching fetal cells from maternal blood. These are very cumbersome, taking several days of highly skilled analytical work. Routine tests are not possible.

A current method involves centrifugation of a blood sample to remove anucleate red cells (the most common cell type in blood) followed by marking of the remaining cells with antibodies. The marked cells are then attracted by antibody reactions to minute magnetic beads which may be separated using a magnetic cell sorting machine. The selected cell population consists mostly of maternal cells but with a very small number of fetal cells.

Variation of this technique commonly termed negative selection, removes most of the maternal white cells (lymphocytes, granulocytes and macrophages) with specific antibodies so that the remainder contains a reasonable proportion of fetal cells. Although such a procedure may give a five to ten fold enrichment of fetal cells, it still leaves the tedious process of visual inspection to find a few required cells among the hundreds or even thousands of irrelevant ones. Such a procedure is too labour intensive for routine use.

According to a first aspect of the present invention diagnostic apparatus comprises: a light transmitting substrate having a specific conjugate immobilised on a surface thereof; means for applying a suspension of cells to the surface; the substrate being adapted to bind cells contained in the suspension having a predetermined characteristic; and the configuration of the surface being arranged to provide a visible density of the bound cells.

The substrate preferably comprises a glass slide, for example a microscope slide. Polymeric substrates for example polystyrene, polycarbonate or polyacrylate may be employed. Such an arrangement facilitates visual inspection of the bound cells.

The configuration of the surface of the light transmitting substrate may be etched or sintered to provide a higher surface area and the conjugate may be coupled directly to this surface. The etched or sintered surface may be treated with an alkylsilane reagent to prevent non-specific cell binding. Use of dimethyl octadecyl chlorosilane or equivalent reagents to create a bonded stationary phase layer is preferred.

According to a preferred aspect of the invention the specific conjugate may be coupled to sub-microscopic beads, the beads being adapted to bind to the substrate. The preferred beads may have a dimension less than 1 micron. Latex beads may be employed, for example onodispersed latex beads prepared by copolymerisation of styrene and a cationic monomer such as vinyl benzyl isothiouronium chloride. Such beads adhere to the hydrophobic monolayer. Coupling by chemical means or physical adhesion may be employed.

The means for analysis may incorporate reagents for detection, extraction, amplification and/or characterisation of chromosomes and DNA in fetal or other cells. The apparatus may incorporate a microscope or automated optical detection apparatus. Bright field, fluorescence or confocal microscopic techniques may be employed.

The conjugates may be immobilised on the substrate surface by pre-treatment (of the substrate, for example a slide) using aminopropyltriethoxysilane (APES) followed by conjugate immobilisation using carbodiimide or an equivalent process. Prepared substrates with immobilised conjugates may be stored in a buffer for several weeks before use.

A test for Downs syndrome using apparatus in accordance with this invention involves microscopic analysis of the slide bound cells following in si tu hybridisation with a DNA probe. Various methods of staining can be used to identify fetal or other cells. These may include histological or immunocytochemical methods. Karyotype analysis may be performed by in si tu hybridisation with appropriate DNA probes. The invention is further described by means of example but not in any limitative sense.

In this example, the immobilised conjugate is SA (secondary antibody) the optimum concentration of which was determined as follows. The optimum SA (secondary antibody) concentration in the Ab immobilisation step was determined as follows. Whole blood cells were washed twice in phosphate- buffered saline (PBS) , labelled with primary mouse anti- glycophorin (GPA) (a red cell specific marker) , washed three times to remove unbound antibody, and resuspended in PBS containing heparin at 0.15 mg/ml . Cells were then applied to an etched slide with secondary antimouse antibodies bound to the surface at the concentrations listed in Table 1 and agitated at low speed on a shaking platform. After washing, slides were stained and cells counted in 10 pseudorandom microscopic fields at 160x magnification.

Table 1

sample [SA (GAM) ] ug/ml mean cell no s . d cells/mm²

GPA 1 0 11 . 1 13 . 7 7xl0³

10 155 153 10⁵

100 3700 540 2 . 3xl0⁶

GPA 2 0 19 . 5 35 . 9 1 . 2xl 0⁴

10 202 190 1 . 3x10^s

100 1207 512 7 . 6x10^s

GAM = Goat Anti-mouse

The slide surface was almost completely covered at 2xl0⁶ cells/mm² and a concentration of 100 ug/ml SA(GAM) gave this practical limit. This represents a 10 - 100 fold increase in cells captured over untreated (0 ug/ml SA(Gam)) slides. Few leucocytes were captured in these experiments.

In a second experiment, SA(GAM) coated slides were used to capture leucocytes from blood samples essentially free of red cells, following their removal by lysis with ammonium chloride. These cells were labelled with either CD45, a pan- leucocyte marker, or CD13, which labels granulocytes and monocytes. Results are shown in Table 2.

Table 2

sample [SA(GAM) ]ug/ml mean cell no s.d eelIs/mm²

CD45 0 0 0 0

50 10.2 6.5xl0³

15.8

CD13 0 0 0 0

10 7.1 4.5xl0³

9.3

100 29.8 1.9xl0⁴

24.5

Of the captured cells in the CD13 labelled sample, 96% were positively identified morphologically as either monocytes or granulocytes, compared to 65% in the starting sample. Any alternative antibody can be substituted for GAM dependent on requirements.

Diagnosis in accordance with the invention may be carried out as follows.

1. Aminopropyltriethoxysilane treated sintered slides were soaked for 2 hours at room temperature in sodium acetate (pH 4.5) containing carbodiimide (20 mg/ml^"1) with constant stirring. The slides were washed gently with several changes of distilled water and dried. A 1 cm diameter circle was marked with a wax crayon. The slides were placed facing upwardly in a humidified chamber, carefully dried around the marked area and excess moisture was removed. The marked area was not allowed to dry out. SA(GAM) solution (50 μl) in sodium acetate buffer (100 μg/ml^"1) was added to the area within the circle and spread carefully and incubated overnight in a refrigerator. The solution was not permitted to dry. Excess antibody was washed off with PBS and the slides were immersed in 100 mM tris buffer containing 1% fully dissolved dried milk powder (blocking buffer) for 30 min. Blocking buffer was washed from the slides and excess moisture was removed from the slides including the area within the marked circle prior to the slides' replacement in the humidified chamber. 2 cm², 5 mm high frames were greased and placed on the slides. A cell suspension, preincubated with primary antibody in a 500 μl volume was rocked for 2 hours at room temperature. The capture targets comprise cells taken from the appropriate region of a Percoll gradient and labelled with primary antibody. The excess antibody was removed by washing the suspension twice with PBS prior to resuspension in 500 μl PBS containing bovine serum albumen (PBS-A) . Excess liquid was decanted from the slides and the frames removed. The slides were transferred to staining pots and washed gently in several changes of PBS. Excess buffer was removed and the slides were fixed and stained. Microscopic analysis of the slides allowed detection of any captured cells, including fetal cells from women known to be carrying a Downs Syndrome fetus.

Slides in accordance with this invention may be manufactured by pre-treatment of glass, polystyrene, polycarbonate or polyperspex slides by exposure to conventional etching reagents in order to increase their surface area and to generate reactive sites for antibody/protein deposition. The treated material may be cut into rectangles having the same dimension as standard microscope slides.

The nature of the substrate may be selected in accordance with the desired sensitivity.

Directly coupled antibodies may be prepared by direct application of monospecific secondary antibodies (SA) , FAb fragments thereof or avidin or streptavidin reagents to the surface of the substrate. Such a substrate will capture relatively abundant cells from a mixed cell population.

An indirectly coupled substrate may be provided by coupling antibodies, FAb fragments or avidin or streptavidin reagents to submicroscopic latex beads which are subsequently absorbed onto the substrate surface. This affords greater sensitivity than directly coupled systems and facilitates a greater range of capture requirements and post-capture processing.

Paramagnetic latex beads may be employed in place of standard latex beads. Such beads may capture cells which have been pre-labelled with a paramagnetically conjugated antibody. Paramagnetic beads find particular applicability where post- capture diagnostic processes may be affected by the capture surface. For example background levels of the coupling antibodies or avidin/streptavidin may interfere with immunocytochemical or cytogenetic procedures.

The required cells may be captured by the latex beads either before or after the beads are adsorbed onto the substrate surface. Premixing of the beads with the cell suspension prior to application to the substrate may be preferred to optimise capture efficiency.

The desired cells may be labelled with monospecific primary antibodies. These antibodies may be either unlabelled for recognition by a secondary antibody layer from the substrate such as GAM, biotin labelled for recognition by a layer of avidin or streptavidin or labelled with microscopic paramagnetic beads for capture by attraction to the magnetic substrate.

The cell mixture, including the labelled cells may be applied to the substrate and gently agitated for a period of up to one hour at 4 to 30^*C. Alternatively the cell suspension mixture may be repeatedly circulated across the active surface in a closed loop. During this time the desired cells are collectively captured by the substrate while unwanted cells remain in suspension. The substrate may be washed to remove these unwanted cells which may in turn be sequentially captured for further investigation by the techniques described above. A succession of substrates adapted to detect different cells may be provided.

Claims

1. Diagnostic apparatus comprising a light transmitting substrate having a conjugate immobilised on a surface thereof; means for applying a suspension of cells to the surface; the substrate being adapted to bind cells having a predetermined characteristic; and the configuration of the surface being arranged to provide a visible density of the bound cells.

2. Diagnostic apparatus as claimed in claim 1 wherein the conjugate is an antibody.

3. Diagnostic apparatus as claimed in claim 2 wherein the conjugate is a monospecific primary or secondary antibody or FAb fragment thereof, a biotinylated reagent, streptavidin or avidin.

4. Diagnostic apparatus as claimed in any preceding claim wherein the conjugate is immobilised directly on the surface of the substrate.

5. Diagnostic apparatus as claimed in any of claims 1 to 3 wherein the cell-type specific conjugate is coupled to sub-microscopic beads, the beads being adapted to adhere to the surface of the substrate.

6. Diagnostic apparatus as claimed in claim 5 wherein the beads are paramagnetic.