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WO2000075647A1 - Systeme et procede de depistage diagnostic prenatal - Google Patents

Systeme et procede de depistage diagnostic prenatal Download PDF

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Publication number
WO2000075647A1
WO2000075647A1 PCT/US2000/015565 US0015565W WO0075647A1 WO 2000075647 A1 WO2000075647 A1 WO 2000075647A1 US 0015565 W US0015565 W US 0015565W WO 0075647 A1 WO0075647 A1 WO 0075647A1
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Prior art keywords
cells
fetal
nrbc
nucleated
maternal
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PCT/US2000/015565
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English (en)
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David W. Sammons
Delia R. Bethell
Wen-Jen Ko
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Bioseparations, Inc.
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Priority to EP00941237A priority Critical patent/EP1203227A4/fr
Publication of WO2000075647A1 publication Critical patent/WO2000075647A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • the present invention relates to a method for characterizing cells using a combination of histochemical staining, antibody labeling and fluorescence in situ hybridization (FISH). More particularly, the present invention relates to a method for characterizing cells using sequential histochemical staining, antibody labeling and FISH to provide data about morphology, antigenic sites as well as ploidy of cells.
  • FISH fluorescence in situ hybridization
  • the present invention also includes an imaging methodology and control software which is useful in routine fluorescent imaging of FISH labeled cells.
  • Fluorescent labels are used as reporter molecules for a number of different applications.
  • Fluorescent fn Situ Hybridization (FISH) techniques are used to detect and identify specific chromosomes and chromosome regions. Gene expression products are identified in whole cells through the use of green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • IMAGESCAN Image Analysis Software System has been used to locate and capture specific fluorescent images while scanning a slide, return to the specific slide location and allow visual confirmation, either immediately or at a later time.
  • One model system is for detection of small fluorescent spots, such as those found with FISH of specific chromosomes.
  • Lymphocytes were separated, centrifuged onto slides and labeled by FISH using X and Y chromosome probes. A set of parameters defining the intensity and size of a fluorescent signal of interest was selected. The slide was automatically scanned. Fields containing images matching the parameter settings were captured. At completion of the scan, captured fields were reviewed and verified for the presence of fluorescent cells. Slides were prepared from peripheral blood and cell nuclei were labeled with DAPI. Parameters were set for the detection of whole cells. Slides were scanned and verified as described above. Final reports were prepared including tables summarizing the number of chromosomes or nuclei captured and an image gallery of captured fields.
  • MS AFP maternal serum alpha-fetoprotein
  • THCG human chorionic gonadotrophin
  • estriol abnormal ultrasonographic findings and family history.
  • CVS chorionic villus sampling
  • amniocentesis amniocentesis and amniocentesis.
  • amniocentesis and CVS have been routinely offered to women who are 35 years of age or older at delivery (Hook EB. "Rates of chromosome abnormalities at different maternal ages," Obstet Gynecol 58, 282-285 (1981)), because the risk of a trisomy (1 in 180) for this age group is about equal to the risk of miscarriage from the procedure (about 1 in 200).
  • CVS has been associated with increased risk for congenital anomalies, specifically transverse limb reduction defects, and oromandibular-limb hypogenesis (Hsieh FJ, et al. "Limb defects after chorionic villus sampling,” Obstet Gynecol Jan 85(1), 84-88. (1987).
  • a method using fetal cells form maternal blood could potentially better identify women at increased risk for fetal chromosome abnormalities and genetic disorders and would be characterized by an improved sensitivity and specificity compared to currently available screening protocols. This would serve as a more accurate protocol for fetal risk assessment and, potentially in the future, prenatal diagnosis.
  • fetal cell types cross the placenta and enter maternal circulation.
  • the cell types that can be isolated from maternal blood for possible use in prenatal diagnostic testing include trophoblasts, lymphocytes, and erythroblasts or nucleated red blood cells.
  • Trophoblasts were the first cells to be identified, but are not suitable for prenatal diagnosis because of their heterogeneous genetic nature and rapid clearance by the maternal pulmonary circulation (Steele CD, et al., "Prenatal diagnosis using fetal cells isolated from maternal peripheral blood: a review" Clin Obstet and Gynecol 39, 801-8136 (1996); Bianchi DS. "Current knowledge about fetal blood cells in the maternal circulation" J Perinat Med 26, 175-185 (1998).
  • Fetal lymphocytes are known to persist in the maternal circulation for 5 years or longer (Ciaranti A., et al., "[Survie de lymphocytes foetaux dans le sang maternal post-parum]" Schwetz Med Klischr 107, 134 (1977); Schroder J, Tiilikainen A, de la Chapelle A. 1974. "Fetal leukocytes in the maternal circulation after delivery: I. Cytological aspects," Transplantation 17, 346 (1974). Post-partum persistence of fetal lymphocytes leads to the potential for diagnostic error arising from genetic analysis of cells originating from a previous pregnancy.
  • Fetal NRBC first appear in the maternal circulation at six weeks of gestation as the liver develops (Hamada H, et al., "Fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age," Hum Genet 91, 427-432 (1993); Liou JD, et al., "Fetal cells in the maternal circulation during the first trimester in pregnancies," Hum Genet 19, 427 (1993).
  • NRBC appear to be best suited for prenatal diagnosis, due to their short half-life in the maternal circulation. Barring recent spontaneous abortion, NRBC should not be present from a prior pregnancy (Elias S, et al. "Isolation of fetal nucleated red blood cells from maternal blood: persistence of cells from prior pregnancy is unlikely to lead to false positive results.” JSoc Gynecol fnvest 3 (Suppl) 359(A) (1996).
  • fetal DNA has been detected in maternal blood using PCR techniques for the measurement of sequences specific to the Y-chromosome.
  • Numbers of fetal cells have been estimated from PCR measurements of fetal DNA in either whole blood (i.e., no enrichment) or samples enriched for fetal cells. Enriching for fetal cells increased the estimated range from 19 - 6,000 to 225 - 22,500 fetal cells per 15 ml of maternal blood (Bianchi DW, Flint AF, Pizzimenti, MF, Knoll JHM, Latt SA. 1990. Isolation of fetal DNA from nucleated erythrocytes in maternal blood.
  • NRBC are short-lived cells of limited replicative capacity, they are ideal candidates for prenatal diagnostic testing. Sekizawa and colleagues (1996) obtained a prenatal diagnosis of Duchenne muscular dystrophy, a single gene disorder, and also were able to accurately determine fetal RhD status.
  • a drawback of the approach is that the gene of interest must be absent from the maternal genome, or the fetal NRBC must be distinguished from the maternal cells.
  • obtaining a full karyotype from fetal erythroblasts has not been reported, expansion of fetal erythroid progenitors has been achieved with cells isolated from the CFS and the sex determined by PCR (Wachtel SS, et al. 1996.
  • fetal cells were identified in one woman; at 9 weeks, in 15/19 of the women; and at 12 weeks, in all of the women.
  • Thomas et al. "The time of appearance and disappearance of fetal DNA from the maternal circulation.”
  • Prenat Diagn 15, 641 (1995) demonstrated that Y-chromosome DNA could be detected in fetal cells as early as 4 weeks, 5 days post-conception (using in vitro fertilization to provide a precise date of conception).
  • fetal genetic material was detected in the maternal circulation by 7 weeks of gestation.
  • Maternal peripheral blood contains both fetal and maternal nucleated erythrocytes (Slunga-Tallberg A, et al. "Maternal origin of nucleated erythrocytes in peripheral venous blood of pregnant women," Hum Genet 96, 53-57 (1994) and Slunga-Tallberg A, et al. "Nucleated erythrocytes in enriched and unenriched peripheral venous blood samples from pregnant and nonpregnant women," Fetal Diagn Ther 9, 291-295 (1995)). Studies by von Eggeling et al.
  • FACS fluorescent activated cell sorting
  • MCS magnetic activated cell sorting
  • CFS charge flow separation
  • the present Applicant BioSeparations, Inc., through its Charge Flow Separation (CFSTM), has shown that fetal cells can be recovered from the excess of maternal cells (Wachtel et al., 1996).
  • the present invention utilizes an integration of three separate technologies for the enrichment, labeling and detection of rare cells into a single system termed the PRENATAL DIAGNOSTI SYSTEM (PDSTM). These technologies consist of the CFS technology, specific detailed protocols for the sequential fluorescence in situ hybridization (FISH and ReFISHTM) of enriched fetal cells, and IMAGESCANTM, a software package that allows the imaging, capture and analysis of labeled cells.
  • FISH and ReFISHTM sequential fluorescence in situ hybridization
  • IMAGESCANTM a software package that allows the imaging, capture and analysis of labeled cells.
  • a successful system for non-invasive prenatal diagnosis must, above all, provide accurate information and be safe for both mother and baby.
  • An accurate diagnosis requires that the maximum number of fetal cells be recovered by the system, and they must be at a high level of purity.
  • the system should also provide rapid, timely information to both patient and clinician, be competitively priced with existing tests, and consist of a user-friendly process which allows operator input for critical steps and decisions.
  • the fetal NRBC must be enriched using the technologies that have the best attributes of recovery and purity possible in order to obtain the maximum sensitivity and specificity for clinical diagnosis.
  • the FISH protocol is performed first, followed by histochemistry/immuno-histochemistry.
  • histochemistry/immuno-histochemistry there is loss of morphological and biochemical features which makes histochemistry/immuno-histochemistry results difficult to interpret.
  • the present invention provides a method for labeling cells using precipitable substrates and histochemical stains and scanning under bright field microscopy to locate labeled cells. Under bright field microscopy, the slides are scanned using IMAGESCAN software (BioSeparations, Inc., Arlington, Arizona) described in greater detail in U.S. Patent No.
  • CHROMAHYB 600 BioSeparations, Inc., Arlington, Arizona
  • DNA probes DNA probes.
  • FISH labeling procedures and the CHROMAHYB 600 reagent are more fully described in co- pending U.S. Patent Applications Nos. 08/949,243, filed October 10, 1997. 08/874,271 filed June 13, 1997, and 08/874,270 filed June 13, 1997 and PCT International Applications Publication Nos. WO 99/19511 published April 22, 1999 and WO 98/56955 published December 17, 1998, the disclosures of each are hereby expressly incorporated by refererence as teaching FISH protocols and hybridization buffers useful with the present invention.
  • IMAGESCAN software automatic repositions the slide to the stored image coordinates corresponding to the labeled cells to view the previously imaged cells for detection of the fluorescent FISH signals.
  • a model system using antibodies specific for subpopulations of leukocytes and FISH for the XY chromosomes demonstrates the utility of the method of the present invention. Potential applications include characterization of fetal cells in maternal cell populations, labeling of cancer cells followed by genetic characterization of the same cells, and monitoring of bone marrow transplants for XY/XX composition following opposite sex transplantation.
  • a system and method for prenatal diagnostic screening is also provided which integrates three major functions: enrichment, labeling and detection. Detection is facilitated by the IMAGESCAN software (BioSeparations, Inc. Arlington, Arizona) which interacts with hardware, including a microscope stage, camera, and optical filters, to scan a sample slide.
  • the IMAGESCAN software creates files which are uploaded onto an internet web server which permits remote access, monitoring and control by any web- based browser.
  • Figure 1 is a flow diagram illustrating the sequential steps of the inventive method for characterizing cells.
  • Figure 2 is a photomicrograph illustrating the use of DAPI as a histochemical label which distinguishes the NRBC from red blood cells by the blue fluorescence of the nucleus.
  • Figure 3 is a bright field photomicrograph illustrating monoclonal antibody labeling of NRBC and some leukocytes with monoclonal antibody 2F6.
  • Figure 4 is a photomicrograph illustrating cells labeled with fluorescent probes for the X and Y chromosomes.
  • Figure 5 is screen shot of the IMAGESCAN software which is used to return to the same slide location and capture corresponding images following different labeling techniques.
  • Figure 6 are graphs representing th effect of varying standard hybridization conditions in the standard FISH protocol.
  • Figure 7 is a screen shot of the FISHFINDERTM software after completion of a scan, illustrating the automatic graphical marking of fields containing cells of interest.
  • Figure 8 is two screenshots of output from the IMAGEFINDER software illustrating data obtained from detection of labeled slides.
  • Figure 9 are screenshots of the FISHFINDER software illlustrating slides prepared with female lymphocytes spiked with 0.1% male cells were scanned in search of SpectrumOrange labeled Y chromosomes. Images from the scan of mixed lymphocytes showing a Y containing cell in the midst of XX cells and the cell counting function of the system. Images are at 40 x using a triple pass FITC/TRITC/UV filter.
  • Figure 10 are screenshots from the FISHFINDER software illustrating cells expressing sgGFPTM (green fluorescent protein) imaged with a FITC filter at 20 x and Rev-sgBFPTM (blue fluorescent protein) imaged with a UV filter.
  • SgGFP and Rev-sgBFP labeled cells were provided by Quantum BioTechnologies, Inc. Montreal, Canada.
  • FIG 11 is a schematic diagram of a Charged Flow Separator (CFS 100 and CFS 200) used in the present invention functionally illustrating operation of the Charged Flow Separator.
  • Figure 12 is a graph depicting the distribution of red blood cells (RBC), white blood cells (WBC) and nucleated red blood cells (NRBC) in fractions eluting from the Charged Flow Separator.
  • RBC red blood cells
  • WBC white blood cells
  • NRBC nucleated red blood cells
  • Figure 13 is a graph illustrating nucleated red blood cell (NRBC) recoveries in samples processed with the CFS 100 and the CFS 200.
  • Figure 14 is a photomicrograph field image and enlarged cell image depicting fluorescently labeled fetal NRBC, fetal RBC, maternal RBC and WBC obtained using the IMAGESCAN system.
  • FIG. 1 is a flow diagram illustrating the method 10 of the present invention and its sequential steps in characterizing cells.
  • the present invention comprises a method 10 which includes a sequence of labeling protocols that allow efficient monoclonal antibody (MoAb) labeling followed by FISH.
  • DAPI (4',6-diamidine-2'-phenylindole dihydrochloride) is used for histochemical labeling of cell nuclei on a slide at step 12. Slides are scanned and images stored with linkage to their location on the slide at step 14. The histochemical label is followed by immunohistochemistry with an indirect monoclonal antibody reaction using an alkaline phosphatase substrate visible by bright field microscopy at step 16. Slides are returned to the microscope, the fields of interest relocated and bright field images captured at step 18.
  • Slides are removed and FISH is performed at step 20. Slides are returned to the microscope for final imaging of the FISH results using fluorescence with the fields selected byased upon the immunohistochemical label being returned to by repositioning the slide using the inventive computer software IMAGEFINDER (BioSeparations, Inc., Arlington, Arizona), at step 22. The data is then analyzed using saved images in an image gallery at step 24.
  • IMAGEFINDER BioSeparations, Inc., Arlington, Arizona
  • the samples on the slides were then treated with DAPI for labeling of DNA in nuclei.
  • the samples were then scanned using fluorescence and a FITC/TRITC/UV filter.
  • slides were scanned by IMAGESCAN TM version 1.0.3 at 1 OOx magnification.
  • the user defines parameters that adjust threshold settings for accurate cell segmentation.
  • Slide is automatically scanned.
  • continuous auto focus maintains optimal focal plane.
  • Objects meeting the segmentation parameters are enumerated and marked.
  • Image data is captured and linked to a color-coded graphic interface. This interface allows the user to retrieve, inspect, save or discard field images. The graphic enables relocation of specific fields after a slide has been removed for further processing. Cells from an image field are selected and saved to a gallery, linked through the graphic interface to the original study.
  • FIGs 2-4 provide different information about the nubleated red blood cells (NRBC).
  • Figure 2 using DAPI as a histochemical label, distinguishes the NRBC from red blood cells (RBC) by the blue fluorescence of the nucleus. The autofluorescence of the surrounding hemoglobin distinguishes NRBC from white blood cells (WBC).
  • RBC red blood cells
  • WBC white blood cells
  • both NRBC and some leukocytes are labeled with the monoclonal antibody 2F6. Thus, NRBC can not be identified using the antibody data alone.
  • Figure 4 the cells have been labeled with probes for the X and Y chromosomes. This provides data indicating which cells are male in origin and which are female. Utilizing data from histochemistry and immunohistochemistry, along with the FISH results, the gender of NRBC and white blood cells is assessed.
  • Figure 5 illustrates the IMAGESCAN software screens used to return to the same slide location and capture corresponding images following different labeling techniques.
  • the IMAGESCAN software enables repeated return to the same cells following sequential labeling methods.
  • the first scan creates a study file with a graphic interface used to facilitate return to selected slide coordinates in subsequent steps.
  • the first scan data is reviewed and fields of interest are marked. At that time, cells within a field can be selected and stored in a gallery linked to the study.
  • the slide is returned to the microscope, the coordinate positions saved in the graphic interface are used to relocate the marked fields.
  • the corresponding cells of interest are selected and saved to the gallery.
  • the same cells can be sequentially labeled using histochemistry, immunohistochemistry and FISH methods.
  • IMAGESCAN software enables the investigator to analyze identical cells, after labeling with different methods, through accurate return to the same fields of interest.
  • Unique information useful for the characterization of cells can be obtained using sequential labeling methods for identification of morphology, protein composition and genomic make up.
  • Peripheral blood lymphocytes were separated from adult male and female anticoagulated blood by centrifugation using Histopaque 1077 and washed twice with phosphate buffered saline (PBS). Cells were counted and male cells were mixed into female cells at approximately 0.1%. Red cells were lysed and nucleated cells were fixed using ice cold Carnoy's (3:1 methanokacetic acid). Slides were processed using the standard CHROMAHYBTM fluorescence in situ hybridization protocol. (BioSeparations, Inc., Arlington, AZ). Slides were scanned using IMAGEFINDERTM consisting of a fluorescent microscope, automated XYZ stage, three chip color CCD camera and FISHFINDERTM software. Standard Hybridization Protocol
  • Scanning parameters are set to count DAPI fluorescent cells and TRITC fluorescent spots.
  • the FISHFINDERTM software automatically provides a graphic marking those fields containing cells of interest. Each field can be reviewed, marked or discarded by working with the saved images or the actual image automatically relocated on the slide.
  • Table 1 The results are set forth in Table 1 , below:
  • Figure 8 are screen shots from the FISHFINDER software, demonstrating the output of information obtained for each spot detected using the FISHFINDER software.
  • Figure 9 are two screenshots from the FISHFINDER software illustrating the results of slides prepared with female lymphocytes spiked with 0.1% male cells were scanned in search of SpectrumOrange labeled Y chromosomes. The system automatically counted and recorded location, size and intensity data characterizing the cells and chromosomes. These data were used to calculate the number of Y chromosomes found as compared to expected. The images from the scan can be transferred into Word, Notepad, Paint or other software for printing. Images from the scan of mixed lymphocytes showing a Y containing cell in the midst of XX cells and the cell counting function of the system. Images are at 40 x using a triple pass FITC/TRITC/UV filter.
  • FISHFINDERTM software used on the IMAGEFINDERTM system provided quantitative results for comparison of assay variables and selection of optimal conditions for the most intense signal.
  • a model system for locating rare cells demonstrated the ability of the FISHFINDERTM software to identify rare signal events. The system is able to select and count specific fluorescent signals based on selected parameters and report the data in tables and images.
  • the present invention provides an imaging analysis tool for comparitive analysis of labeled cellular microscopic images and comparing immunohistochemical labels with fluorescence labels to yield an accurate and rapid scan of a sample for labeled cells. Additionally, the present invention provides a method for characterizing cells using a combination of histochemical staining, antibody labeling and fluorescence in situ hybridization (FISH).
  • FISH fluorescence in situ hybridization
  • PDS Prenatal Diagnostic System
  • the PDS protocol consists of three major tasks: enrichment, labeling and detection.
  • Enrichment of the sample requires three discrete steps: 1) density gradient centrifugation; 2) Charge Flow Separation (CFS); and 3) magnetic positive selection of NRBC and lysis of mature maternal RBC.
  • Labeling requires two additional steps: 4) fixation and slide preparation and 5), fluorescence in situ hybridization (FISH).
  • FISH fluorescence in situ hybridization
  • the detection of fetal cells is the final task, and uses the IMAGESCAN program to 6) scan and 7) capture the labeled NRBC and its chromosomes.
  • Centrifugation is the first step in the enrichment process, and used to remove the bulk of the platelets and mature RBC without the loss of NRBC. Unfortunately, the losses during the centrifugation can be substantial, but this is balanced by a significant 150-fold increase in purity. Our current method yields about 56 ⁇ 30%> recovery of NRBC.
  • the second step in the enrichment process is charge flow separation.
  • the CFS technology is an adaptation of free-flow electrophoresis, using a flow of buffer opposing the electrical charge to enhance the separation of cells. Briefly, the sample enters from the bottom of the separator, and is move in an upward direction by the flow of buffer. An electrical charge moves across the separator from the anode to the cathode, counterbalanced by a second flow of buffer from the cathode to the anode. Unwanted fluid gradients which would minimize separation are controlled by the use of stabilization media. In this system, cells are separated based on their charge-to-mass ratio.
  • the CFS separator geometry is shown schematically in Figure 1 1, which depicts a CFS separator having a separation chamber sub-divided into a plurality of separation channels divided by membranes.
  • a sample is input into a channel, an electrical field is applied, and a buffer flow having an orientation vector opposing the electrical field, known as a buffer counterflow is applied to the sample.
  • a plurality of fractions elute from the separation chamber containing the separated sample.
  • RBC have the highest electrophoretic mobilities, and WBC the lowest.
  • NRBC in maternal circulation originate in the fetal liver and represent various stages of development in the erythroid pathway.
  • the NRBC are both RBC-like and WBC-like in their surface properties. Electrophoretically the NRBC should separate under the anodal shoulder of the RBC peak and under the cathodal shoulder of the WBC peak, depending upon its stage of erythroid maturation.
  • Figure 12 illustrates a typical distribution profile of NRBC in relation to the RBC and WBC profiles separated with the CFS. Under these conditions, approximately 70%> of the NRBC are located in CFS fractions 6 and 7 along with about 15% of the WBC and about 50% of the RBC.
  • NRBC is the fetal cell found in maternal blood preferred for non-invasive prenatal diagnosis
  • BioSeparations, Inc. has focused its research methods on this cell type.
  • the early literature suggested a frequency of fetal cells in maternal circulation on the order of 1 per 10 6 to 10 8 .
  • a frequency of 1 fetal cell per 10 maternal cells would represent 75,000 fetal cells in 15 ml of maternal blood.
  • most of the current technologies should be capable of isolating and identifying fetal cells.
  • the number of fetal cells per maternal cell is more on the order of 1 per 10 to 10 , or less.
  • the CFS was used to purify NRBC that were subsequently subjected to FISH analysis for the Y-chromosome (Wachtel et al., 1996; Wachtel et al., 1998).
  • fourteen maternal samples were processed with the CFS, Carnoy's fixed and labeled with DNA probes to the X- and Y-chromosomes. Male gender was correctly identified in nine cases (90%>) when 10,000 nuclei were scored. Because the true starting concentration of fetal cells in maternal cells was unknown, an inclusion criteria of at least 0.2% (1 NRBC/500 nucleated cells) was selected.
  • the range of NRBC recovered from the CFS was 380 - 36,000 (mean 10,319). Assuming 30 - 35% of the NRBC are fetal (von Eggeling et al., 1997; Wachtel et al., 1996), then an average of greater than 3000 fetal cells were isolated in the samples analyzed.
  • CFS is 799, the number of WBC at this step is unacceptably high to allow deposition of all the cells on 1-2 slides. This necessitates increasing both the recovery and purity of the fetal cells in order to approach a level of detection of 100%.
  • BioSeparations. Inc. the focus has been to increase the recovery of the NRBC. To that end, improvements in the CFS and IMAGESCAN, the cornerstones of the PDS system, have been made.
  • the PDS method steps were characterized and optimized for recovery of NRBC, for final purity of NRBC in relation to the contaminating maternal leukocyte and mature RBC, and for the identification of fetal cells.
  • a model system to mimic maternal blood was utilized. Samples were prepared by mixing a small amount of neonatal cord blood with peripheral blood from an adult of the opposite sex. Typically, 50,000 to 300,000 NRBC were spiked into 15 ml of the adult blood. The spiked sample with the fetal NRBC at a known concentration was processed with each PDS step being analyzed for recovery and purity.
  • NRBC number was evaluated by counting benzidine-stained NRBC under brightfield illumination.
  • the CFS-200 provides a 3-fold increase in recovery of NRBC as compared with the CFS- 100, as shown in Figure 13. This should translate into a similar increase in recovery of fetal NRBC from maternal blood to be performed in this study.
  • the third step in the enrichment process is magnetic positive selection and lysis of contaminating mature RBC and WBC.
  • all cells need to be contained on a single slide.
  • a single slide requires four hours to scan and detect fetal cells.
  • the number of nucleated maternal cells is still an issue for timely analysis of samples with very low numbers of fetal cells.
  • Preliminary data indicate the desired purity can be obtained after the CFS with the use of monoclonal antibodies to NRBC. Treatment with a monoclonal antibody cocktail conjugated to paramagnetic fluid would result in minimal loss of fetal cells (10%>), but a significant increase in purity, the maternal cell number being reduced one log (data not shown).
  • the use of monoclonal antibody positive selection on the CFS fractions containing over 70% of the fetal cells has significant advantages over the same technique applied to density gradient processed blood.
  • Selection is done on 2-3 ml of sample with total cell number of approximately 100 million and white cell number of 3-4 million.
  • the CFS combined with density gradient centrifugation has increased the purity of the fetal cells by 300-fold or more. Since none of the antibodies identified to date are completely specific for the fetal NRBC, there is less probability of cross-reactive sites interfering with the blocking the antibody.
  • the combination of CFS and magnetic positive selection will enable the sensitivity of detecting fetal cells in the circulation of pregnant women to approach 100%.
  • Carnoy's fixation and cell lysis removes the contaminating maternal RBC, allowing all cells to be deposited on a single slide. Preliminary studies indicate approximately 80% > recovery of NRBC after fixation and slide preparation.
  • Fixation and slide preparation improvements have contributed to the increased recovery of NRBC. Modifications in protocols, decreasing centrifugation times and reduction in the number of steps have been made. Slide coatings have been compared and a standard procedure for cleaning and coating slides developed. These adjustments have decreased losses during fixation and slide preparation.
  • Modification of the ChromaHybTM hybridization buffer has improved both hybridization efficiency and signal intensity following FISH.
  • CFS fractions were depleted of WBC using CD45 monoclonal antibody to remove leukocytes.
  • the hybridization efficiency with X-and Y-DNA probes of the NRBC enriched fraction was 96.6 ⁇ 4%.
  • the mean hybridization efficiency of four samples was 96 ⁇ 2%.
  • FISH optimization also resulted in increased signal intensity. The increased intensity allows a more accurate determination of hybridization efficiency and use of a lower magnification, which decreases analysis time.
  • IMAGESCANTM is a software package developed by BioSeparations, Inc. that aids in the detection of cytologic features during microscopic investigations.
  • the software interacts with hardware, such as the stage, camera, filters, and nosepiece, during successive scans of a sample slide. During a scan, the software will adjust, both automatically and interactively with the user, hardware settings such as stage location and camera exposure time to facilitate the acquisition of cellular images of the highest possible quality.
  • IMAGESCAN analyzes that image to locate cytologic features, such as cellular boundaries and chromosomes. The results of these analyses are stored in tabular form and are available to the user.
  • IMAGESCAN also creates files that are placed on a web server so that studies can be monitored remotely via any web browser. When a study is completed, all data, images, and results associated with a study are automatically archived to disk. The user can review all analyses performed automatically by IMAGESCAN as well as further analyze the original images and related data, as desired.
  • the centrifugation and CFS steps yield an estimated recovery of 55 - 70% and 66%, respectively, of the starting number of NRBC.
  • the use of monoclonal antibody positive selection allows for the further purification of the sample and achieves fetal cell recoveries of at least 90%> of the NRBC in the CFS fractions. Recovery of approximately 80% of the NRBC is observed with the fixation step.
  • FISH and IMAGESCAN each have a sensitivity of 95.5%.
  • Table 3 presents data demonstrating the depletion of contaminating maternal RBC and WBC and enrichment for NRBC during the progressive steps of the PDS.
  • the post- lysis step results in a significant increase in purity with the removal of the mature WBC .
  • 80%> of the NRBC remain and can be centrifuged onto a single slide.
  • Minimal losses of less than 10% are experienced during the FISH and IMAGESCAN steps.
  • Initial cell numbers are based on Documeta Geigy (Diem and Lentner, 1970).
  • Table 4 shows an estimated recovery of fetal and maternal cells by PDS beginning with 15 milliliters of maternal blood and different ratios of fetal to maternal cells. These data are based on the estimates of the number of fetal cells which cross the placenta into maternal circulation (Simpson and Elias, 1993; Bianchi et al., 1990).
  • the frequency of 1/10 7 and 1/10 8 is commonly quoted as the expected range of fetal cells in maternal blood. Given that fetal cells have been detected in 50 - 80% of pregnant women (de la Cruz, 1998; Holzgreve et al., 1998b) one can predict that the fetal cells may be more rare than expected. Our data suggest that we can detect fetal cells in maternal blood at a frequency of 1 in 10 9 maternal cells, or as low as 18 fetal cells in the final step of processing. This sensitivity is consistent with detecting the range of fetal cells previously estimated from the study in Table 2. Using the data that 35% of NRBC in maternal blood are fetal in origin, it is estimated that as few as 40 fetal cells could be isolated with the PDS and detected on a single slide with IMAGESCAN.
  • the Prenatal Diagnostic System (PDS) of the present invention is carried out utilizing different fluorescent labels to cells for the detection of different potential targets.
  • probe or set of probes such as the fluorescent ⁇ -hemoglobin mRNA probe of Step 7, is applied to the samples.
  • the cells are imaged, the fluorescent labels are removed, new labels are applied, and new cell images are obtained.
  • the results are analyzed and reported during Step 13 of the Analysis/Reporting Task.
  • the data are analyzed and reported in Step 20.
  • the sequential steps employed in the PDS are the following:
  • the blood is centrifuged on a Ficoll gradient for 40 minutes.
  • the plasma and the erythrocyte portions are discarded and the remaining cells are washed twice.
  • the cells are then resuspended in CFS running buffer containing 1% BSA to a final concentration of 3xl0 7 cells/ml and a maximum of 10 ml is processed through the CFS instrument.
  • Cell counts are performed using a Z2 Coulter Particle Count and Size Analyzer.
  • the CFS unit is operated under computer control with user-defined input at decision points. On startup, the unit is automatically flushed with water and electrolyte buffer. A solution of CFS running buffer with 0.5% BSA is added through the sample inlet port to condition the flowpath of the cells. The cells, which have been resuspended in CFS running buffer with 0.5% BSA, are loaded onto the instrument and the computer program is activated. The cells are pumped into the separation chamber, separated and collected into discrete fractions. When the run is complete, the CFS is prepared for the end of day shutdown. The cell fractions are centrifuged, and the cell pellets are then resuspended in the appropriate buffer prior to counting.
  • the CFS system has a network interface card (NIC) installed that enables the system to be connected to the Internet. While the CFS control program is running, the CFS current and voltage information will be sent as a Transmission Control Protocol/Internet Protocol (TCP/IP) packet to a web server, located at BioSeparations, Inc. in Arlington. The current and voltage information will be graphed onto an X-Y plot and displayed on a continuously updated web page. BioSeparations, Inc. personnel can then monitor the status of the CFS system via an Internet browser. This would allow the early detection of any separation problems indicated by abnormal current and voltage fluctuations.
  • TCP/IP Transmission Control Protocol/Internet Protocol
  • Selective Lysis Fetal NRBC are further enriched from contaminating maternal RBC by selective lysis of adult RBC using a modification of the method of Boyer et al. "Enrichment of erythrocytes of fetal origin from adult-fetal blood mixtures via selective hemolysis of adult blood cells: an aid to antenatal diagnosis of hemoglobinopathies," Blood 47, 886-897. (1976).
  • the cells are applied to a slide using cytocentrifugation and are fixed in 3: 1 ethanol/methanol .
  • the probe to detect mRNA for the ⁇ F globin chain of the fetal hemoglobin (HbF) can be used as an additional marker for fetal NRBC during FISH, and in some cases can be used to identify NRBC from female fetuses.
  • ImageScan systematically scans the entire sample disk of fluorescently labeled cells, field-by-field, using a 20x objective and a 2.5x internal lens located in the camera mount of the digital camera. This gives an overall magnification of 50x for the digital field images.
  • the resolution of a 50x digital image approximately corresponds to that of a 200x optical image as seen by a human user. At this magnification, approximately 1050 fields are required to cover a sample disk with a 12mm diameter.
  • the scan proceeds field-by-field, moving horizontally and vertically across the entire area of the sample disk. An image of each field, referred to as a "field image", is captured as the scan proceeds. Parameters used in the scanning process, such as exposure time, are set at the beginning of the scan, based upon the characteristics of the sample and the desired objectives of the scan.
  • ImageScan scans the disk as part of Step 8, it automatically searches the sample for ⁇ Hb mRNA-labeled NRBC, each of which exhibits fluorescence in the cytoplasm.
  • NRBC exhibit a red fluorescence in their cytoplasm as seen through a tetramethyl-rhodamine-isothiocyanate (TRITC) filter.
  • TRITC tetramethyl-rhodamine-isothiocyanate
  • NRBC are identified automatically by ImageScan through the combined presence in one cell of red fluorescence in the cytoplasm and blue fluorescence in the nucleus of a single cell.
  • the majority of the fluorescing NRBC are expected to be of fetal origin. However, a small percentage of maternal NRBC may also be labeled in the samples.
  • An image of the potential NRBC is captured automatically and stored into an image gallery.
  • the field-by-field progress of the scan is displayed on password-protected web pages on the Internet through a web server located at BioSeparations, Inc. in Arlington. Image and data files are transferred from remote imaging sites to the web server in Arlington as each scan proceeds. All information related to the ongoing scan, including field and cell images, is available to a remote user via the Internet. These web pages will be used by scientists at BioSeparations, Inc. to monitor the scanning process in Arlington and to provide feedback to the local users at remote sites.
  • ImageScan For each field in the scan during Step 8, ImageScan performs the following steps: i) Position the slide so that the field is under the microscope objective, ii) Focus the image, iii) Switch to the TRITC filter. iv) Identify cells expressing the ⁇ -Hb gene within their cytoplasm. This is done by searching the field image for circular reddish objects. Note that it is generally accepted that 1-4% of maternal RBC express the ⁇ -Hb gene, so maternal as well as fetal RBC will be identified in this step. The pattern recognition algorithms used by ImageScan might also identify some undetermined contaminants (false positives) in this step. v) Switch to the DAPI/TRITC filter.
  • the photo-micrographs in Figure 14 show fluorescently labeled fetal NRBC, fetal RBC, maternal RBC, and WBC.
  • the figure illustrates the reddish color of the cytoplasm within the fetal cells as compared to maternal RBC, which lack fluorescently labeled ⁇ - hemoglobin mRNA.
  • the expanded image (i.e. cell image) to the right represents the tile image that ImageScan captures automatically during scanning of fields and subsequent gallery generation.
  • the green dot within the blue DAPI-stained nucleus of the fetal NRBC is a Y-chromosome that has been labeled with FISH.
  • the user has several options to help identify true NRBC.
  • the user can instruct ImageScan to automatically relocate the cell on the slide, then examine the cell under higher magnification or through different filters. Images of cells that the user has determined not to be NRBC will be removed from the gallery of cell images; these cells will not be imaged during subsequent steps (i.e., Steps 1 1, 15, and 18).
  • An intuitive graphic-user interface has been developed to facilitate the review process.
  • Step 9 a revised gallery of cell images, chosen from the images obtained in Step 8, is produced with each image containing one or more true NRBC. This revised gallery is available for remote monitoring via the Internet at BioSeparations, Inc. and elsewhere. 10. FISH (X-, Y-probes)
  • the previously used slide is subject to a "ReFISH" procedure, in which the hybridized sample is washed in a 1 :9 dilution of ChromaHyb Washstock and dehydrated in a series of ethanol washes (Thompson et al., 1997).
  • the slide is air-dried and submitted to the ChromaHyb Pretreatment procedure which includes a 70% acid incubation to lyse fetal red blood cells and remove interfering cytoplasm for the following hybridization with directly labeled DNA probes for the X- and Y-chromosomes.
  • the procedure itself is carried out according to manufacturers' instructions, as well as posthybridization washes in 1 :80, 1 :200 and 1 :400 dilutions of the ChromaHyb Washstock. Before visualization the slides are counterstained with DAPI.
  • Step 11 the slide containing the cells with hybridized fluorescent X- and Y- probes is repositioned on the stage.
  • the X-chromosome probe is a strongly fluorescent red label while the Y-chromosome probe is a more weakly fluorescent green label.
  • ImageScan automatically positions the slide so that the cell is centered under the objective.
  • a cell image is then captured using a triple-pass FITC/TRITC/DAPI filter at 20x magnification.
  • a new gallery of cell images is obtained as the cells are relocated and imaged with the X- and Y-chromosome probes.
  • the stage can be repositioned so that the cell in question may be examined directly on the slide.
  • Higher magnification objectives and different filters can be selected, as appropriate, for review of the images.
  • the weaker, green Y-probe may be more clearly seen through the more selective FITC/DAPI dual-pass filter instead of the FITC/TRITC/DAPI filter.
  • the results of this review and those of Step 9 will be analyzed and report as part of Step 13.
  • Slides are relabeled with different fluorescent DNA probes in Step 14 in order to study the ploidy of autosomal chromosomes 13 and 21. 13.
  • the primary endpoint is the determined gender of the fetus.
  • the statistic used to assess the performance of the PDS system for predicting fetal sex is the overall agreement proportion of PDS answers which agree with the amniocentesis or CVS gender determination. This proportion is accompanied by the 95% confidence interval estimate for agreement proportion, computed with exact binomial (Clopper-Pearson) methods.
  • the slide is submitted to another ReFISH procedure by using the wash protocol developed by Thompson et al. "New rapid FISH techniques using X and Y 13/21, 18 chromosome synthetic DNA probes," In Vitro Cell Devel Biol-Animal 33, 31A. (1997).
  • Several authors were able to show that sequential FISH analysis can be performed between 3 to 9 times on the same slide without loss of specificity and intensity of the hybridization signals. (Wang et al., 1995; Dierlamm et al., “Successful use of the same slide for consecutive fluorescence in situ hybridization experiments,".
  • Step 15 the slides containing the cells with hybridized fluorescent 13- and 21- probes are repositioned on the stage. Both of these probes are red and will be located on separate slides.
  • each cell in the revised gallery from step 9 is automatically centered under the objective and imaged at 20x magnification with the TRITC/DAPI, dual-pass filter. Only the red channel will be stored for each image.
  • a new gallery of cell images is obtained from each slide as the cells labeled with either the chromosome- 13 or the chromosome-21 fluorescent probe are relocated and imaged. 16.
  • FISH FISH (18- robes)
  • the slide is submitted to the ReFISH procedure, by removing formerly bound DNA probe in a 1 :9 wash dilution of ChromaHyb Washstock (Thompson et al., 1997).
  • the slide is hybridized with a directly labeled DNA probe for chromosome 18 according to manufacturers' conditions and visualized after the posthybridization washes by DAPI counterstain. The evaluation of the slide progresses as outlined in step 18.
  • Steps 11 and 15 a new gallery of cell images is obtained from each slide, as the cells labeled with the chromosome- 18 fluorescent probe are relocated and imaged.
  • the review process in Step 19 is similar to that of Steps 12 and 16. However, in this case, cells are labeled with a single red probe for chromosome 18.
  • a TRITC/DAPI dual-pass filter will be used for review. If necessary, higher magnification or use of a single-pass TRITC filter can be used to aid in recognizing the probes. Results of this review will be analyzed and presented in a final report during Step 20.
  • the prevalence occurrence rates of monosomy, diso y (expected result except for X and Y) and trisomy determinations for each of the chromosomes (X, Y, 13, 18 and 21) will be computed among the 180 subjects in this part of the study. Any noted occurrences of additional polysomies beyond 3 copies will be tabulated. Mosaicism rates will be computed, and Clopper-Pearson confidence intervals for the occurrence rates will be established. The results will be compared to those obtained from the corresponding amniocentesis or CVS results, and an overall agreement rate computed. The occurrence rate of false positives and negatives will also be determined based on this comparison, with the same corresponding 95% confidence intervals determined.
  • an integrated system of cell enrichment, labeling and detection which permits users to image labeled cells using computer-controlled automated imaging, image analysis to detect and store labeled cells coordinates on each slide, and to permit internet-based access and remote control.

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Abstract

L'invention concerne un dépistage diagnostic prénatal utilisant un système comprenant l'enrichissement, le marquage et la détection des cellules. Le marquage caractérise les cellules à l'aide d'une combinaison de coloration histochimique, de marquage et d'hybridation in situ fluorescente d'anticorps. Le marquage peut également être réalisé au moyen d'un procédé de détection de cellules marquées par marquage immunohistochimique ; il permet de comparer le positionnement des marques immunohistochimiques par rapport à une marque d'hybridation in situ fluorescente préalablement détectée.
PCT/US2000/015565 1999-06-04 2000-06-05 Systeme et procede de depistage diagnostic prenatal WO2000075647A1 (fr)

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US11647743B2 (en) 2002-10-16 2023-05-16 Streck Llc Method and device for collecting and preserving cells for analysis
US11634747B2 (en) 2009-01-21 2023-04-25 Streck Llc Preservation of fetal nucleic acids in maternal plasma
US10689686B2 (en) 2009-02-18 2020-06-23 Streck, Inc. Preservation of cell-free nucleic acids
US9657227B2 (en) 2009-02-18 2017-05-23 Streck, Inc. Preservation of cell-free RNA in blood samples
US20180216165A1 (en) 2009-02-18 2018-08-02 Streck, Inc. Preservation of cell-free nucleic acids
US11761025B2 (en) 2009-02-18 2023-09-19 Streck Llc Preservation of cell-free nucleic acids
US10144955B2 (en) 2009-02-18 2018-12-04 Streck, Inc. Methods for preservation of cell-free nucleic acids
US10294513B2 (en) 2009-02-18 2019-05-21 Streck, Inc. Preservation of cell-free nucleic acids
US9926590B2 (en) 2009-02-18 2018-03-27 Streck, Inc. Devices and compositions for preservation of cell-free nucleic acids
US8586306B2 (en) 2009-02-18 2013-11-19 Streck, Inc. Preservation of cell-free RNA in blood samples
US9956281B2 (en) 2011-05-04 2018-05-01 Streck, Inc. Inactivated virus compositions and methods of preparing such compositions
EP2726871A4 (fr) * 2011-06-30 2015-03-04 Univ Singapore Détection de cellule sanguine rouge nucléée f tale
US10674721B2 (en) 2013-07-24 2020-06-09 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
US11547111B2 (en) 2013-07-24 2023-01-10 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
US10091984B2 (en) 2013-07-24 2018-10-09 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
US12114654B2 (en) 2013-07-24 2024-10-15 Streck Llc Compositions and methods for stabilizing circulating tumor cells
US11168351B2 (en) 2015-03-05 2021-11-09 Streck, Inc. Stabilization of nucleic acids in urine
US11299764B2 (en) 2015-11-20 2022-04-12 Streck, Inc. Single spin process for blood plasma separation and plasma composition including preservative
US11506655B2 (en) 2016-07-29 2022-11-22 Streck, Inc. Suspension composition for hematology analysis control

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