WO1994002830A1

WO1994002830A1 - Analysis of cells

Info

Publication number: WO1994002830A1
Application number: PCT/GB1993/001561
Authority: WO
Inventors: Melvyn Greaves; Catherine Mary Price; Susan Colman
Original assignee: The Institute Of Cancer Research
Priority date: 1992-07-23
Filing date: 1993-07-23
Publication date: 1994-02-03
Also published as: GB9215710D0

Abstract

A process for the phenotyping and genotyping of a cell which comprises (i) fixing a cell to a solid support; (ii) contacting the cell with an antibody, which antibody is labelled, either before or after being brought into contact with the cell, with a histochemical stain which stain is capable of fluorescing; (iii) contacting the cell with a DNA probe, which DNA probe is labelled, either before or after being brought into contact with the cell, with a colorimetric label which fluoresces at a different wavelength from the histochemical stain.

Description

ANALYSIS OF CELLS

The present invention relates to the genetic screening of cells, eg cells of cancer (including leukemic) or pre-natal (fetal) lineages.

It is often desirable to examine cells simultaneously for a particular phenotype and a corresponding genotype. For example, the correlation of cell type and chromosomal abnormality can provide additional information relating to lineage involvement and diagnosis in leukaemia and the developmental level of the clonal leukemic cells which would be the target for any therapy.

Generally, it has not until now been possible to identify both the phenotype (by antibody probing) and genotype (by DNA hybridization) of a cell simultaneously by conventional histochemical methods.

Recently, Kibbelaar et al (Blood, 29(7); 1823-1828, (1992)) have found that a cell can be stained in solution with an antibody probe attached to a fluorescent label and that a cell thus stained can be further stained with a DNA probe attached to a different fluorescent label, allowing simultaneous identification of a phenotype and a genotype.

From a practical point of view, the technique disclosed by Kibbelaar has a number of disadvantages. The procedures involved in contacting cells with antibodies and conducting in situ hybridization with DNA probes are both tasks which require technical expertise. Such expertise is not always available in pathology laboratories who perform only routine analysis of cells of patients, but rely on other, fewer, centres for more complicated analysis of such samples. However, the technique of Kibbelaar must be performed on fresh cell samples because the cells are contacted with the labelled antibody in solution.

SUBSTITUTESHEET Further, once the labelled antibody has been contacted with the cells being analyzed, the entire procedure described by Kibbelaar must be performed as soon as possible, due to the fact that the fluorescent stains fade, and become undetectable within a few weeks or days.

A further disadvantage of the need to perform rapid analysis of cells is that this does not allow for historical analysis of the cells at a later date. For example, it may be desirable to determine the results of a first phenotyping experiment before deciding which antibodies should be used on a second phenotyping experiment. It may also be the case that as new antibodies become available and the understanding of diseases increases, information can be obtained from samples taken from patients who have long since died.

Thus, although Kibbelaar et al show that cells can be analyzed by combined immunotyping and genotyping, the method disclosed has limited practical application as a routine clinical technique.

The present invention thus provides a process for the analysis of a cell which comprises (i) contacting a cell with an antibody, which antibody is labelled, either before or after being brought into contact with the cell, with a colorimetric label; (ii) contacting the cell with a DNA probe, which DNA probe is labelled, either before or after being brought into contact with the cell, with a different colorimetric label, characterised in that the cell is fixed to a solid support prior to labelling.

By fixing the cell to a solid support, eg. a glass slide, the cell sample can be stored for a considerable length of time prior to conducting the reaction with the antibody and DNA probes. This would allow the cells to be obtained in any pathology laboratory and subsequently analyzed in a specialist analytical laboratory elsewhere where there is the requisite level of

SUBSTITUTE SHEET expertise at cell labelling techniques. This would allow the cells to be analyzed one or two weeks, or a month, or even a year or more after they have been removed from a patient. It would also allow a first sample of cells to be analysed and a second sample, taken at the same time, to be analysed with different antibody and/or DNA markers which are selected in the light of the result of the first analysis.

A further advantage of the present invention is that by fixing the cell on a solid support, the cell membrane is made permeable, which allows the contents of the cell cytoplasm to be available for labelling. This will increase the signal obtained from cells positive for the marker being analyzed, which will help distinguish such positive cells from mere background signals.

The term "colorimetric" refers to two types of label. These are histochemical markers and fluorescent markers. Histochemical stains are permanent whereas, as described above, fluorescent stains fade after relatively short periods of time.

It has recently been found that the histochemical stain, alkaline phosphatase-Fast Red, is also capable of fluorescing but does not, unlike conventional fluorescent stains, fade.

The present invention thus further provides a process for the phenotyping and genotyping of a cell which comprises (i) contacting the cell with an antibody, which antibody is labelled, either before or after being brought into contact with the cell, with a colorimetric label; (ii) contacting the cell with a DNA probe, which DNA probe is labelled, either before or after being brought into contact with the cell, with a different colorimetric label, characterised in that the colorimetric label used to label the antibody is a histochemical stain capable of fluorescing.

Preferably, the histochemical stain is alkaline phosphatase-Fast

SUBSTITUTESHEET Red. Other histochemical stains include fuschin.

By using such a histochemical stain, the cells can be stored after step (i) above for any desired length of time prior to staining with a DNA probe.

Preferably, the phenotyping and genotyping of cells is conducted in a process which comprises (i) fixing a cell to a solid support; (ii) contacting the cell with an antibody, which antibody is labelled, either before or after being brought into contact with the cell, with a histochemical stain which stain is capable of fluorescing; (iii) contacting the cell with a DNA probe, which DNA probe is labelled, either before or after being brought into contact with the cell, with a colorimetric label which fluoresces at a different wavelength from the histochemical stain.

We have found that the above process provides all the advantages of long term storage and ease of handling mentioned above together with the advantages of enhanced signal strength from cells being analyzed. These advantages allow cell samples to be collected from different collection centres, each of which may be too small or too inexperienced to provide the capability to immunotype and genotype cells, and for these cell samples to be sent to a central diagnostic service, which can process all the samples together, thus reducing costs and increasing reliability of diagnosis.

Thus, the present invention also provides a method for the phenotyping and genotyping of a cell fixed to a solid support which comprises (i) contacting the cell with an antibody, which antibody is labelled, either before or after being brought into contact with the cell, with a histochemical stain which stain is capable of fluorescing; (ii) contacting the cell with a DNA probe, which DNA probe is labelled, either before or after being

SUBSTITUTESHEET brought into contact with the cell, with a colorimetric label which fluoresces at a different wavelength from the histochemical stain.

The steps of contacting the antibody to the cell sample, and labelling the antibody are known in the art per se. Usually, an unlabelled antibody produced in a first species (eg mouse) directed against an antigen of interest is contacted with the cells. A second antibody against the antibody of the first species is used to detect the first antibody, and this second antibody is either labelled, or capable of being labelled by a colorimetric stain. Variations on this are known in the art and can be used in the present invention.

Similarly, a DNA probe may be labelled with a first marker, suitably biotin. The first marker can be detected with a further probe, eg. fluoresceinated avidin. Variations of this are again known in the art (eg amplification of the avidin by biotinylated anti-avidin) and may be used in the present invention.

As used herein, the term "DNA probe" means a probe capable of detecting genomic DNA in a cell. The probe will usually comprise DNA. However, the probe may also comprise modified bases or even comprise RNA. In order that the probe may be detectable, it will usually comprise at least one base which is suitably labelled, eg with biotin. The precise nature of the DNA probe may be determined by those of skill in the art, taking into account the nature of the target DNA and reagents available for the probe target.

Preferably, the cell is contacted with the antibody prior to being contacted with the DNA.

In the embodiments of the present invention described above, the contacted cell is usually exposed to radiation of a wavelength

SUBSTITUTESHEET that will cause both the labelled antibody and the labelled probe to fluoresce. The result of the fluorescence may be recorded in any manner desired, eg by photography or by digitisation and recordal in a computer. The latter method may be advantageous when the two wavelengths at which the probe and antibody stains fluoresce appear similar to the naked eye, since the digitised record can be manipulated and represented by the recordal system in other ways, eg with "false colours".

The invention may be used to examine cells, eg human cells or other mammalian cells, in situations where information on cell lineage and genotype are required simultaneously. This is useful in the study of cancers, especially leukemias and preleukemic conditions, eg polycythemia vera. Such diseases are often manifested by a clonal lineage of cells which have an abnormal chromosome content (eg trisomies, deletions or translocations) and aberrant expression of cell markers. Information concerning the nature of such markers will assist the physician in the specific diagnosis of the disease, to monitor the progress of the disease and to develop improved therapies for the treatment of the disease, eg by the use of toxic antibodies directed against markers expressed by the leukemic cells, or by monitoring cell numbers after treatment with different drugs.

The invention can also find utility in examining fetal cells for prenatal diagnosis. For example, chorionic villus sampling (CVS) , which can be conducted in the first trimester of pregnancy, is in theory preferable to amniocentesis as a method of prenatal diagnosis, which is conducted in the middle of the second trimester, since late termination of pregnancy (in the event of the detection of a genetic abnormality) is generally undesirable for both medical and social reasons. However, CVS is a difficult procedure and may result in samples which contain a mixture of fetal and maternal cells. Such samples will be

SUBSTITUTESHEET difficult to analyze by conventional methods. By using the present invention, it is possible to label the cells with a fetal antibody marker and conduct an examination for genomic abnormalities, eg trisomy 21 or genetic defects inherited in a normal mendelian fashion. Examples of such defects include muscular dystrophy and cystic fibrosis.

Fetal nucleated erythrocytes have also been isolated from maternal blood during pregnancy using monoclonal antibodies and flow sorting¹⁶"²¹ and have been detected as early as 11 weeks of gestational age^16,19,20. Analogous techniques may be used to obtain fetal cells from maternal blood using a Magnetic-Activated Cell Sorter (MACS) , using a suitable MALS column (eg available from Milteny Biotec GmbH) .

The following example illustrates the invention.

EXAMPLE 1.

Two patients with the myeloproliterative disease polycythemia vera (PV) were studied, using trisomy of chromosome 8 as the marker for the abnormal clone. Trisomy 8 is a non-random chromosome abnormality in PV occurring in approximately 16% of karyotypically abnormal cases and in common with other chromosomal changes (eg. del(20q) and +9) it may occur alone or as a secondary event.

Brief details of the two patients used in this study are given below. Conventional cytogenetic analysis revealed that the peripheral blood from both patients was chimeric for normal and abnormal karotypes. In both patients the abnormal clone or clones had trisomy of chromosome 8 in addition to other abnormalities. This trisomy was used as a marker for the malignant cells.

SUBSTITUTESHEET Patient 1. This 67 year old man presented in October 1991 with a scrotal hydrocele. He was noted to have a raised haemoglobin and further investigations, including the production of erythropoietin independent erythroid colony growth, confirmed the diagnosis of PV. Cytogenetic analysis showed a normal male karyotype and the presence of two hyperdiplid clones both with trisomy 8 in addition to other chromosomal abnormalities: 46,XY/47,XY,+8,+9, -12,t (6;12) 48,XY, +8,+9, 12,t (6;12) ,del (12q) . Peripheral blood samples were obtained from this patient at this time. Shortly after presentation he required emergency surgery for an ischaemic lower limb and sustained a fatal cerebro- vascular event post-operatively.

Patient 2. This 60 year old lady presented with PV in 1978 from which time she received intermittent therapy with busulphan and venesections. In 1989 her blood film was noted to show red cell morphology consistent with marrow fibrosis and this was confirmed with marrow biopsy. Six months before this study a repeat marrow examination showed progressive fibrosis and also features suggestive of developing leukemic transformation. Cytogenetic analysis showed a normal female karyotype and a hyperdiploid clone with trisomy 8 in addition to other abnormalities: 46,XX/48,XX+8,+del (l)qter-p21) ,t (l;21)ql2;pll) . Peripheral blood samples were obtained from the patient for this study in June 1991 when the presence of the same hyperdiploid clone was confirmed. In August 1991 she died following a short illness with hemorrhagic features.

Cell Material. Peripheral blood samples from both patients and a normal female control were obtained by venesection and placed in preservative free heparin. Whole peripheral blood was cultured from 2 to 48 hours and harvested for routine cytogenetic analysis. The remainder of the sample was centrifuged over Lymphoprep (Nycomed, Oslo, Norway) to obtain a mononuclear cell (MNC) fraction. The MNC were used in hemopoietic colony assays,

SUBSTITUTESHEET cytocentrifuged on to glass slides or stored in liquid nitrogen.

Colony assay. MNC were cultured at a concentration of 2 x 10⁵/mL in Iscove's medium containing 40% fetal calf serum, 2% bovine serum albumin (BSA) , 10^"4M 2-mercaptoethanol, 1% methylcellulose, 5 units of erythropoietin and 30 units GM-CSF. Cultures were incubated at 37oC in a humidified 5% C0₂ atmosphere for 14 days. Individual erythroid (BFU-E) and granulocyte/macrophage (CFU-GM) colonies were removed using a micropipiette, suspended in phosphate buffered saline (PBS) and cytocentrifuged onto glass microscope slides. The slides were either fixed in methanol and Giemsa stained to confirm morphological cell type or air dried, wrapped in foil and stored at -20oc for later staining and FISH.

Slide preparation. Fresh MNC from patient 1 and a normal control, and cryopreserved MNC from patient 2 were resuspended in PBS to a final concentration of 10⁶/mL. lOOμL of cell suspension were cytocentrifuged onto glass slides (cytospins) , air dried and stored, wrapped in foil, at -20oC.

Immunophenotype analysis. Cytospins of MNC were stained with a panel of murine monoclonal antibodies to determine cell lineage using a three stage (APAAP) unlabelled bridge method for immunophenotyping¹. Briefly, slides stored at -20°C were defrosted at room temperature before being unwrapped, fixed in acetone and air dried. Following rehydration in Tris buffered saline (TBS) the slides were incubated in a moist chamber with 20μL of normal human serum (NHS), diluted 1:10 in TBS, at room temperature for 5 minutes. lOμL of the appropriate primary antibody (murine monoclonal antibody) was added and the slides incubated as above for 1 hour. The slides were washed in a TBS for 20 minutes and 20μL of the second layer, an unlabelled rabbit anti-mouse antibody (DAKO Ltd, High Wycombe, UK) , was added and the slides incubated for 45 minutes in the humid chamber. The slides were washed in TBS as above. 20μL of anti-rabbit APAAP

SUBSTITUTE SHEET complexes (DAKO Ltd) were then added and the slides were again incubated at room temperature for 45 minutes. The slides were washed as before and the alkaline phosphate Fast Red substrate was added. Colour development was monitored using a light microscope. Finally the slides were washed in TBS for 10 minutes and the cells counterstained in Hematoxylin. The slides were then air dried, mounted in an aqueous mounting medium Glycergel (DAKO Ltd) and scored for positive cells using a light microscope.

Antibodies. Lymphoid associated; T-cells CD3 (T28; a gift from

Dr P Beverley) , B-cells CD22 (Leul4; Becton Dickinson, Mountain

View, CA) . Myeloid associated: Granulocytes/monocytes CDllc

(3.9; a gift from Dr N Hogg), immature granulocytes/monocytes

CD13 (MY7; Coulter Electronics Ltd, Luton, UK), monocytes CD14 (MY4 : a gift from Dr J Griffen) and megakaryocytes CD41 (J15; a gift from Dr A McMichael) . Hematopoietic progenitors: CD34 (QBEND10; Quantum Biosystems, Cambridge, UK) . Erythrocytes: Anti-glycophorin A (AGA) R10; a gift from Dr P Edwards) .

Fluorescent in si tu hybridization. Cytocentrifuged cells from both colony and MnC cells (following the APAAP technique) and standard cytogenetic slide preparations were treated using the same in si tu hybridization protocol. In preliminary experiments we found that chromosome specific centromeric repeat probes (for chromosomes 1, 4, 8 and X) all gave strong signals in interphase nuclei from cytospun MNCs (data not shown) . We concluded that for efficient hybridization of these probes pretreatment, with the consequent loss of morphological integrity, was not necessary. Cytogenetic slides were stored for up to 2 weeks at room temperature before hybridization. APAAP preparations were kept at room temperature for up to 2 months before hybridization without any obvious deterioration in the hybridization signal. Shortly before hybridization the cover slips were removed in hot water and the slides allowed to air dry. Cell preparations were

SUBSTITUTESHEET heat denatured in 2X SSC/70% formamide at 70oC for 2 minutes, dehydrated through 70%, 90% and 100% alcohol and air dried at room temperature. Hybridization buffer (50% formamide/10% dextran sulphate in 2X SSC pH 7.0) containing 2ng/μL of biotin labelled probe [chromosome 8 specific alpha-satellite probe D8Z1

(Oncor Science, Gaithersburg, MD) ] was denatured at 70oc for 7 minutes and cooled on ice at 4°c. 15μL of the hybridization solution was added to each slide under a sealed coverslip and incubated overnight in a moist chamber at 37oC. The coverslips were removed in 2X SSC and the slides washed in 50% formamide/2X

SSC (pH 7.0) at 45°c for 40 minutes, twice in 2X SSC (pH 7.0) for

5 minutes at room temperature and once in IX SSC (pH 7.0) for 5 minutes at room temperature. Slides were stored at room temperature in phosphate buffer (0.1M NaH₂P0₄,0.1M Na₂HP0₄ pH8.0)

Detection. The slides were blocked in 3% BSA in 4X SSC at 37oC for 10 minutes. 200μL of fluoresceinated avidin (Vector Laboratories, Burlingame, CA) , at a concentration of 5μg/mL in 4X SSC containing 5% low fat milk and 0.05% Triton X, was placed on the slide under a coverslip and incubated for 40 minutes at 37oc. The signal was amplified using biotinylated anti-avidin

(Vector) at a concentration of 5μg/mL and a further layer of fluoresceinated avidin. Between detection steps the slides were washed twice for 5 minutes in 4X SSC/0.05% Triton X pH 7.0.

Following a final wash in phosphate buffer the cells were mounted in fluorescence antifade (Citifluor, Nottingham University, UK) or antifade containing the counterstains propidium iodide (3ng/μL) or DAPI (diamidino-2-phenyl-indole dihydrochloride, 200ng/mL) (both Sigma, Poole, UK) . Double labelled fluorescent cells were analysed using either a Zeiss photomicroscope III equipped with epifluorescence and a dual band-pass filter (Omega, Brattleboro, VT) or a Bio-Rad MRC-600 laser scanning confocal microscope equipped with akrypton/argon ion laser (488/568nm line excitation with dual channel, 522nm and 585nm, emission filters) . Cells viewed on the Zeiss photomicroscope were photographed on

SUBSTITUTESHEET Kodak Ektar 400 colour film (Eastman Kodak, Rochester, NY) . Confocal images were printed on Sony Mavigraph video print film (Sony Corporation, Japan) . RESULTS In si tu hybridization. In si tu hybridization with the chromosome 8 specific probe confirmed the presence of the trisomy in cytogenetic preparations from the peripheral blood cells of both patients.

The false negative rate for the probe was assessed by scoring 200 cells APAAP control slides (immunoglobulin sub-class specific) from the normal control (Table 1) . In common with other centromeric repeat probes the false negative rate (1 signal per cell) for the chromosome 8 probe is higher (2-5%) than the false positive rate (3 signals per cell) at less than 1% in the normal control. Scorable cells with no signal contributed to less than 0.5% of the total cells observed. Cells with three signals in APAAP control slides from patients 1 and 2 were observed in 21% and 71% respectively (Table 1) . In addition, patient 1 had a small percentage 3%) of cells with 4 signals.

Colony analysis. Colonies from patient 1 and 2 showed the presence of the trisomy in both myeloid (1/2 and 13/13) and erythroid (3/3 and 3/3) hematopoetic progenitors (Table 2) . The number of cells analysed per colony and the percentage of cells showing 1, 2 or 3 chromosome 8 specific signals are given in Table 2. The hybridization efficiency in colony cytospin cells is reduced so that the. number of informative cells is lower in comparison with normal MNC cytospins. In consequence the false negative rate for centromeric probes in this type of preparation is higher at 5-17% whereas the false positive rate remains similar to that observed in normal MNC cytospins (2-2.5%) . This problem however, is offset by the clonal nature of the preparations whereby only a few cells in the colony need be scored to confirm the genotype.

SUBSTITUTESHEET Immunophenotype analysis. Results of the immunophenotyping of the MNCs for both patients are given in Table 3. Patient 1 was positive for the following antibodies: CD3(9%), CD14(7.5%), CDllc(87%), CD22(2.5%) and CD34 (2.5%) . No megakaryocytes (J15) or nucleated red cells (RIO) were observed. Patient 2 was positive for the erythroid antibody RIO (48%) although faint staining was seen on several cells using the myeloid (MY7) and T-lymphocyte (T28) antibodies. Morphological staining of the latter showed that the cytoplasm was damaged which would account for the diffuse cytoplasmic staining. Frozen MNCs from other patient samples (stored for up to 8 years and not detailed in this report) produced suitable intact cells for APAAP/FISH suggesting that patient sample variation rather than storage method was responsible for the cell damage.

In si tu and immunophenotype analysis. In si tu results with the chromosome 8 probe on the immunophenotyped cells are given in Table 4. Erythroid (RIO) positive cells only could be scored for patient 2 and of these the majority (90%) had three signals confirming the involvement of the erytroid lineage in the neoplastic clone. An R10+ cell photographed before and after FISH illustrated the dual fluorescence signal obtained in antibody positive cells. B- and T-lymphocytes from patient 1 showed a normal two signal distribution of chromosome 8 whereas monocytes and granulocytes showed two separate populations; 63%- 76% with two signals and 21%-35% with three signals. A small proportion of CDllc+ cells (1%) had 4 signals. These cells either represent a third clone with two additional copies of chromosome 8 or are polyploid cells resulting from aberrant mitosis. A mixed population of normal (46%) and trisomic cells (64%) were also observed in the CD34+ cells from patient 1.

Example 1 shows it is possible to analyse both phenotype and genotype simultaneously in the same interphase cell. Two major advantages of this method are fist that it allows a direct cell

SUBSTITUTESHEET by cell analysis of lineage and chromosomal markers and negates the need for sequential photography followed by relocation of specific cells, and second that the number of analysable cells is high due to the absence of harsh pretreatment steps which damage cytoplasmic and cell surface features. This technique has been used successfully to show discordant lineage involvement of the neoplastic clone in the myeloproliferative disease polycythemia vera.

In both patients studied the clonal marker (trisomy 8) was present in early myeloid (CFU-GM) and erythroid (BFU-E) cells. In patient 1 trisomy 8 was present in a subset of mature myeloid cells (CDllc÷, CD13+, CD14+) but was absent in all B- and T- lymphocytes scored. The CD34+ cells from this patient included both normal and trisomic populations and since CD34+ cells include both lymphoid and myeloid progenitors^2,3 we assume that the trisomy is most likely to be myeloid restricted.

Previous studies of hematopoiesis in patients with PV using glucose-6-phosphate dehydrogenase (G-6-PD) , phosphoglycerate kinase (PGK) or hypoxanthine phospribosyl transferase (HPRT) X linked polymorphisms have shown clonality of erythrocyte, granulocyte and platelet lineages^4"6, whole blood⁶, and blood leukocytes^7,8. In a recent study of PGK polymorphism using PcR

(Polymerase chain reaction) amplified DNA from different cell populations⁹ from two patients with PV were shown to have clonal granulocytes and BFU-E, whereas in a third patient the granulocytes were polyclonal but most of the BFU-E expressed the same PGK allele implying a clonal derivation. These studies provide convincing evidence of clonality of leucocytes and myeloid/erythroid cells in most, but not all, cases of PV. In addition, a study of clonality in PV using PGK polymorphisms has shown that whole separated blood granulocytes were clonal in 4 cases assessed but T-lymphocytes from the same patients were polyclonal⁷. This result could indicate that the 'target' cell

SUBSTITUTE SHEET for clonal selection in PV is not the lymphoid/myeloid stern cell but a myeloid lineage committed cell. Alteratively, the longevity of T-cells and the slow rate of new T-cell production in adults could obscure a clonal contribution to the lineage. Rare cases of ly phoblastic transformation of PV ^10,11 as in T-cell blastic crisis of CML^12,14 indirectly implicate the lymphoid/myeloid stem cell. A detailed study of Epstein-Barr virus-transformed B-lymphoblastoid lines from a patient with PV has provided evidence of clonality of the B-lymphoid lineage thus indicating this disease can involve a stem cell pluripotent for both the lymphoid and myeloid series¹⁵. In our study trisomy 8 could not be found in either B- or T-lymphocytes. This observation may be a true reflection of the lineage restriction of the disease in patient 1 or could be a consequence of the trisomy failing to fully define the myeloproliferative clone. Similar arguments apply to those myeloid cells in patient 1 with only two copies of chromosome 8. The incidence of clonal chromosomal aberrations in PV at diagnosis or preceding cytotoxic therapy is relatively low (13-17%) ; it is possible therefore that trisomy 8, although a non random finding in PV, occurs as a secondary event in an already established neoplastic clone. Studies to determine clonality of specific cell lineages using HPRT/PGK polymorphism in combination with the dual immunophenotype and chromosomal marker analysis presented in this report should provide additional information on the lineage relationships of cells involved in the initiation and progression of PV, other myeloproliferative disorders and leukemia.

EXAMPLE 2

Blood samples from 6 pregnant women were obtained and the blood enriched for fetal nucleated erythrocytes. Three blood samples from non-pregnant females were used as controls.

The cells -were immunotyped and genotyped using the methods

SUBSTITUTESHEET described in Example 1 above, but using as antibody probe the mouse IgGl anti-fetal hemoglobin antibody UCHγ²², which specifically binds to fetal erythrocytes, and using as the DNA probe GMGYIO, a chromosome Y specific repeat probe²³. Cells which were negative for GMGYIO were reprobed with DXZl, a chromosome X specific repeat^24,25.

No UCHγ postive nucleated cells were detected in the 3 control samples. The results of the 6 test samples are shown in Table 5. In the 5 samples which proved informative, the results obtained were in exact agreement with fetal sex determination by cytogenetic analysis of amniotic fluid samples (CAAF) .

SUBSTITUTESHEET TABLE 1. In situ hybridization signal for the chromosome 8 specific probe in MNC cytospin slides*

Number of signals (% from 200 cells) 1 2 3 4

Control 2 97.5 0.5 Patient 1 5 71 21 3 Patient 2 3 26 71

- : no cells with 4 signals observed

*: Slides treated with APAAP control reagents (see text;

SUBSTITUTESHEET TABLE 2. Frequency of in situ hybridization signals of the chromosome 8 specific probe in BFU-E and CFU-GM colonies.

TABLE 3 Frequency of APAAP positive cells in MNC cytospins

Lymphoid associated Erythroid Precurso

CD3 CD22 AGA CD34 (T28) (Leu 14)

(RIO) (QBEND10

87 ND 7.25 2.5 2819 400

ND 48 680

-: No positive cells observed

ND: Not done

AGA: An i glycopho in A

* : Damaged cytoplasm

TABLE 4 Frequency of In situ hybridization signals of the chromosome 8 specific probe in immunophenotyped cells

Total No. Number of signals (%) Antibody Cells Scored 1 2 3 4

Patient 1

Lymphoid associated CD3(T28)

CD22 (Leul4)

Myeloid associated CDllc(3.9)

CD14 (My4)

Precursor CD34 (QBEND10) 100 0 46 64

Patient 2

Erythroid AGA(R10) 100 8 2 90

SUBSTITUTE SHEET TABLE 5 Details of six blood samples from pregnant women stuided by combined immunostaining and genotyping.

CD

C CD

H C H m * Three HbF positive cells, which were both Y & X hybridisation negative, were in the middle of a cell clump and overlapped by other cells.

CO I m ** One of the HbF positive cells was covered by other cells and could not be analysed by m

H FISH.

REFERENCES

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SUBSTITUTESHEET

Claims

1. A process for the phenotyping and genotyping of a cell which comprises (i) fixing a cell to a solid support; (ii) contacting the cell with an antibody, which antibody is

_Λ labelled, either before or after being brought into contact with the cell, with a histochemical stain which stain is capable of fluorescing; (iii) contacting the cell with a DNA probe, which DNA probe is labelled, either before or after being brought into contact with the cell, with a colorimetric label which fluoresces at a different wavelength from the histochemical stain.

2. A process according to claim 1 wherein the cell is fixed to a solid support more than one week prior to bringing the cell into contact with an antibody.

3. A process for the phenotyping and genotyping of a cell fixed to a solid support which comprises (i) contacting the cell with an antibody, which antibody is labelled, either before or after being brought into contact with the cell, with a histochemical stain which stain is capable of fluorescing; (ii) contacting the cell with a DNA probe, which DNA probe is labelled, either before or after being brought into contact with the cell, with a colorimetric label which fluoresces at a different wavelength from the histochemical stain.

4. A process for the phenotyping and genotyping of a cell which comprises (i) contacting the cell with an antibody, which antibody is labelled, either before or after being brought into contact with the cell, with a colorimetric label; (ii) contacting the cell with a DNA probe, which DNA probe is labelled, either before or after being brought into contact with the cell, with a different colorimetric

SUBSTITUTE SHEET label, characterised in that the colorimetric label used to label the antibody is a histochemical stain capable of fluorescing.

5. A process according to any one of claims 1 to 4 wherein the histochemical stain is alkaline phosphatase-Fast Red.

6. A process for the phenotyping and genotyping of a cell which comprises (i) contacting a cell with an antibody, which antibody is labelled, either before or after being brought into contact with the cell, with a colorimetric label; (ii) contacting the cell with a DNA probe, which DNA probe is labelled, either before or after being brought into contact with the cell, with a different colorimetric label, characterised in that the cell is fixed to a solid support prior to labelling.

7. A process according to claim 6 wherein the colourimetric label to which the antibody is attached is a histochemical stain which is capable of fluorescing such as alkaline phosphatase-Fast Red.

8. A process according to any one of claims 1 to 3, 6 or 7 wherein the solid support is a glass slide.

9. A process according to any one of the preceding claims which further comprises exposing the contacted cell to radiation of a wavelength that will cause both the labelled antibody and the labelled probe to fluoresce.

10. A process according to claim 9 which further comprises recording the result of the fluorescence by photography or by digitisation and recordal in a computer.

SUBSTITUTESHEET

11. A process according to any one of the preceding claims in which the cell is a white blood cell.

12. A method for the diagnosis or prognosis of cancer which comprises a process according to any one of claims 1 to 11.

13. A method according to claim 12 for the diagnosis or prognosis of leukemia or preleukemia.

14. A process according to any one of claims 1 to 10 wherein the cell is a fetal cell.

15. A method for the prenatal detection of genetic abnormalities in a foetus which comprises a process according to claim 14.

16. A process according to claim 14 wherein the DNA probe is a chromosome 21 probe.

17. A method for the pre-natal detection of Down's sydrome which comprises a process according to claim 16.

18. A process according to claim 14 wherein the DNA probe is an X chromosome or Y chromosome specific probe.

19. A method for pre-natal determination of sex which comprises a process according to claim 18.

20. A kit for the phenotyping and genotyping of cells which comprises an antibody labelled with a histochemical stain which stain is capable of fluorescing and a DNA probe which is suitable for labelling by a colorimetric label which fluoresces at a different wavelength from the histochemical stain.

SUBSTITUTESHEET

21. A kit according to claim 20 for use in a process or method according to any one of claims 1 to 19.

SUBSTITUTE SHEET