Payperview combined binary ratio fluorescence in situ hybridiziation cobra-fish development and appl


The most remarkable one is the test for deletion or duplication of the subtelomeric regions leading to a clinical picture mostly characterized by multiple congenital anomalies and intellectual disability. Being time-consuming and expensive to evaluate chromosomal rearrangements in the whole genome by FISH led to the development of new techniques such as array-based comparative genomic hybridization [ 33 — 35 ]. Comparative genomic hybridization CGH which is based on competitive hybridization of amplified tumor DNA and normal DNA hybridized on normal metaphase slide was first developed by Kallioniemi et al.

If we look at the evolution of genetic technologies, the emergence of new technologies has always been inevitable due to the necessities revealed by the previous technologies. Although hybridization-based methods, which allow screening RNA, DNA, or protein such as northern blot, southern blot, or western blot, respectively, are widely used, innovative and powerful microarray hybridization methods were developed in the s. To summarize briefly, in this method, DNA of the patient is labeled with a specific color green and mixed with exactly the same amount of DNA of a normal control, which is labeled with a different color red.

This DNA mixture is then hybridized to the denaturated probe DNA on the glass and signal intensity ratios of test over reference are measured. Yellow dye appears when both patient and reference DNA are equal in proportion because of the presence of the same amount of red and green dyes, while regions with copy number losses are visualized as red and gains are green.

This technique permits the detection of whole genome copy number variation CNV duplications and deletions at high resolution [ 36 ]. Array-CGH failed to detect the recessive disease genes, mosaic aneuploidy, uniparental disomy UPD , or heterochromatic rearrangements.

These arrays have the highest resolution of all of the available array-based platforms. Combination of array-CGH and SNP genotyping in a single platform increases the clinical diagnostic capability and uncovers the detection of small copy number variants [ 39 ]. The major drawback of array-based CGH is that it can only detect unbalanced rearrangements and is unable to detect balanced aberrations such as chromosome translocations, inversions, and insertions.

However, recently, a modified array protocol, called translocation CGH tCGH , was developed to address recurrent translocation breakpoints [ 40 ]. Larger genomic changes such as deletions, duplications, and translocations can be detected by conventional karyotyping, FISH, or array-CGH methods but single nucleotide changes cannot be detected by these techniques.

Molecular genetic techniques were rapidly developed after the establishment of polymerase chain reactions that enabled generating thousands to millions of copies of a particular DNA sequence [ 41 ].

Automation of PCR was greatly facilitated and simplified the detection of genomic mutations. SSCP and RFLP, the most widely used techniques for mutation screening method in genetic diagnostic laboratories, were not able to detect every mutation, so development of new methods was needed.

If the sequence of the gene of interest is not known, it may be difficult to interpret the results of these techniques. The determination of DNA sequencing enabled identifying the definite nucleotide changes in the targeted genes. This necessity was overcome by Maxam and Gilbert introducing Maxam-Gilbert chemical sequencing technology based on chemical modification of DNA followed by cleavage at specific bases [ 43 ].

Despite the efficiency of Maxam-Gilbert sequencing method, the use of hazardous chemical and inability to read long PCR fragments made this method replaced by Sanger sequencing that was based on dideoxynucleotide chain termination [ 44 ]. Manual Sanger sequencing method has been improved by the introduction of first generation of automated DNA sequencers [ 45 ]. Automatization of DNA sequencing enabled sequencing human genome in a fast and accurate way.

With the advances in the field of molecular genetics, it became possible to launch the Human Genome Project to reveal the complete human genome.

The programme was launched in the USA with an effort of the Department of Energy and the National Institutes of Health in collaboration involving 20 groups in One day later, in parallel with HGP, Craig Venter who launched a human genome sequencing project by Celera Genomics using shot-gun sequencing method published the whole human genome sequence in Science [ 47 ]. Human Genome Project not only revealed the complete sequence of the human genome but also led to a huge improvement in the sequencing technology.

Amplification of the gene of interest in the affected individual s enabled revealing mutations associated with specific monogenic disorders. Although automation of traditional dideoxy DNA sequencing Sanger method increases the efficiency of DNA sequencing, it was still not cost- and time-effective.

A new technology called massively parallel sequencing MPS erasing these disadvantages was developed by Lynx Therapeutics [ 49 ]. This technology using reads of multiple reactions simultaneously and generating large amounts of sequence data in parallel provided a large impetus for exome sequencing, whole genome sequencing, and transcriptome and methylation profiling.

NGS technology is widely used for a variety of clinical and research applications, such as detection of rare genomic variants by whole genome resequencing or targeted sequencing, transcriptome profiling of cells, tissues, and organisms, and identification of epigenetic markers for disease diagnosis. One of the most successful applications of NGS technology is genome-wide discovery of causal variants in single gene disorders and complex genomic landscapes of many diseases. While whole genome or whole exome sequencing is the most comprehensive strategy in the diagnosis of unknown diseases and identification of new disease genes, targeted sequencing using selected panels of genes can reduce the sequencing time and cost by combining the diseases in the same group or pathway genes in known clinical pictures such as intellectual disability, neurometabolic disorders, or malignancies [ 50 ].

In addition to cost-effective advantages, sequencing the small part of the genome allows reducing the number of variations that in turn reduce the cost and time needed for data interpretation [ 51 ]. Targeted sequencing opened a new window in the diagnosis of several diseases with unknown etiology. Following the rapid advances in NGS technologies, the role of NGS in routine clinical practice will increase exponentially.

Noninvasive prenatal diagnosis by using NGS is another application of this new technology. The most important step in the prenatal diagnostic procedures is obtaining fetal material to evaluate genetic condition. For years, invasive and noninvasive tests have been used to assess the fetal health, particularly chromosomal abnormalities, during the pregnancy.

Noninvasive tests measure epiphenomena, which does not analyze the pathology underlying the clinical picture of interest. Their sensitivity and specificity have not also reached the expected level despite several studies.

On the other hand, invasive tests have been found to be associated with significant risks for both the mother and fetus. The identification of cell-free fetal DNA in maternal circulation and analyzing this fetal material by using NGS opened up a new horizon in the field of reproductive medical care. Despite main advantages of NGS technology, the researchers and clinicians still have many concerns about the implementation of NGS in practice [ 52 ].

The interpretation of huge amount of data obtained by NGS technology, billing and insurance issues, duration and content of consent process, and disclosure of incidental findings and variants of unknown significance were the main challenges related to offering this technology [ 53 ]. Approximately 10 years ago, karyotyping was the gold standard in patients with intellectual disability but array-CGH analysis has become the first line diagnostic test replacing karyotyping and FISH nowadays.

As evident from this example, approaches to the genetic-related diseases could change in parallel with the advances in technology and science. In , a small chromosome called Philadelphia chromosome was identified to be the cause of the chronic myeloid leukaemia CML. It was shown in by the chromosome banding technique that this chromosome was a result of a translocation between chromosomes 9 and 22 [ 54 , 55 ].

The following studies revealed that this fusion gene resulted in activation of a tyrosine kinase, which led to the discovery of a tyrosine kinase inhibitor drug Gleevec that was shown to be a highly successful treatment for CML [ 57 ]. Genetic test that will be used in the diagnosis should be chosen very carefully, which might not be the newest or the most sophisticated one. Sometimes only a karyotype could be enough to identify the genetic condition in the patient instead of more complicated array-CGH or NGS methods.

As the technology in genetics rapidly evolves, new insights in terms of data interpretation and genetic counseling including pretest counseling, return of results, and posttest counseling need to be considered.

Databases and consortium reports regarding the experiences of the clinicians and geneticists are crucial for integration of genomics into clinical practice. If the developments in genetics and computer technologies continue to progress at their current speed, history has shown us we can look forward to some amazing developments in human life in the very near future.

Some realistic scenarios of human life in the future could even see us carrying identity cards, which include our genome characteristics, rather than the format we are currently using.

Gene corrections, cloned individuals and organs, and even genetic-based techniques as a primary laboratory analysis in almost all human diseases for a clinician will no longer be a dream. We have come to the point nowadays where genetic testing is commercially available; the individual now has the possible means to access this delicate information named as direct to consumer DTC genetic testing.

Today, more than 25 companies, from all over the world, offer DTC service to the public. Serious concerns, however, regarding the use of this kind of service, have been raised in terms of misleading and incidental results derived from unproven or invalidated data. Moreover, there is also a significant risk for unauthorized use of sensitive genetic information by big business, particularly in the fields such as health insurance.

On the other hand, DTC does provide early awareness of genetic diseases and thereby offers individuals the opportunity to play an active role in their own health care. The issue at stake here leads us to the same difficult medical ethics dilemma: To conclude, in parallel with the rapid developments in the field of genetic technologies, ethical and legal issues regarding the implementation of those technologies need to be addressed.

Background of the Invention Human cervical epithelium can undergo premalignant morphologic changes which have been termed cervical dysplasia or cervical intraepithelial neoplasia CIN. Higher degrees of dysplasia have increased probability of progressing to invasive carcinoma of the cervix. Natural history of cervical intraepithelial neoplasia: Pathology of early cervical neoplasia. Church Hill Livingston, Approximately 60 years ago, Dr. George Papanicolaou developed the so called Pap smear as a method of screening women for cervical dysplasia.

The scraped cells are then smeared onto a slide which is immediately fixed by an alcohol based solution. The cells fixed onto the slide are stained Pap stain and subsequently analyzed under the microscope. Features such as nuclear size, nuclear chromasia, nuclear irregularity, and koilocytic changes allowed the examiner to identify dysplastic cells. More recently the Bethesda system of reporting has fine tuned the morphologic criteria for identifying dysplastic cervical cells using the Pap smear.

Springer-Veralg New York Inc. This gave rise to the categorization of dysplastic cells as either low grade squamous intraepithelial lesions LSIL or high grade squamous intraepithelial lesions HSIL. These in turn correspond to low grade dysplasia and high grade dysplasia respectively. The introduction of the Pap smear revolutionized cervical screening. This has made the Pap smear test the most successful cancer screening test ever employed, thus having become the standard for cervical cancer screening.

However, the Pap smear does have its limitations. These begin almost immediately as the sample is taken. First, only a small portion of the sample collected from the patient is transferred to the slide during the smearing of the spatula collecting device, contributing to the false negative results. The specimen in the spatula is immediatel 'smeared onto a slide and sprayed with an alcohol based fixative.

Both of these steps frequently produce artifacts precluding a definitive diagnosis. If the specimen is smeared too strongly onto the slide, cells will be crushed and destroyed rendering inadequate morphology for diagnosis. Also, if more than a couple of seconds elapse from the time the spray fixative is applied, the cells will display air drying artifact.

This is evidenced by artificially enlarged cells with pale nuclei, which also often preclude lesional diagnosis. The Pap smear relies on human observer interpretation. This introduces human error as well as interoperative variability as a source of error. False positive results, on the other hand, may be as high as Due to the possibly of false positive results a repeat follow-up Pap smear or immediate colposcopy is recommended subsequent to a positive result.

Symptomatic generated repeat testing is often the result of advanced disease such as invasive carcinoma. Medicare pays for screening paps once every three years.

Therefore for at least this population it is likely that at least three years may pass before the false negative is identified. These issues make the review of a pap smear one of the most labor intensive, least paid, and most litigious specimen that is. In the last 5 to 10 years several technologies have been developed in an effort address the limitations of the Pap test. The first and most widely accepted of these has been the so called ThinPrep pap test developed by Cytec corporation several years ago.

It was the first of two FDA approved automated alternatives to the preparation of cervical scrape specimens. The method was revolutionary as it was the first time that a liquid based specimen transport media and monolayer slide preparation was used for cervical specimen diagnosis.

The ThinPrep pap test methodology is basically as follows. The collecting device is then immediately submerged in a ethanol based transport media and swirled a number of times, approximately 10, in order to dislodge the cells.

The sample is then placed in the ThinPrep machine where the fluid is suctioned through a semi permeable membrane by means of applying negative pressure. The membrane has microscopic pores that let fluid through but prevent the cells from passing. The negative pressure is applied in pulses required to suction a defined volume of fluid. The time interval required for the suction pulse to obtain a defined fluid volume is also monitored.

As more cells adhere to the membrane, resistance requires subsequently longer suction pulse intervals to obtain the defined fluid volume. Once the suction pulse time interval reaches a predetermined threshold value all suction ceases. The predetermined threshold time interval would be one that was judged to give the greatest cellularity without having cells on top of each other or a monolayer of cells.

The cells on the membrane are then transferred to a glass slide with the aid of slight positive pressure in the direction opposite to the suction. The cells now adhered to the slide are stained by the Papanicolaou method and cover slipped for microscopic examination. There are several reasons for the increased acceptance of the ThinPrep pap test in recent years. Virtually the entire sample is collected in the preservative fluid. There is a randomized, representative transfer of cells onto the glass slide.

There is an even distribution of cells onto the slide minimizing obscuring material, cellular clumping, and cellular overlap. The immediate transfer of cells onto a fluid environment eliminates mechanical and air drying artifact rendering improved morphologic detail for diagnosis. Kabawat SE, et al. As with the Pap smear test there are certain limitations with the ThinPrep pap test that make it less than ideal. As with the Pap smear test, this is a labor intensive method of slide preparation followed by human microscopic examination.

The ability to judge cells by the company they keep is lost due to the so called cellular randomization of the specimen. This can be particularly problematic when trying to identify populations of atypical immature squamous metaplastic like cells that may actually be HSIL cells or associated with such. Yearbook of Pathology and Laboratory Medicine The remainder of the cells are left in the preservative fluid. The ThinPrep pap test also comes at an increased cost.

These include all of the costs of a conventional Pap screen in addition to ThinPrep specific costs. This does not include the additional cost incurred should the slide have an abnormality that would require review by a pathologist. An alternate FDA approved automated method of preparing cervical scrape specimens for analysis has been devised by Tripath Corporation via the Autocyte machine.

The cellular material is dislodged into the preservative liquid. The cellular material is then centrifuged into a pellet. The cellular pellet is resuspended in concentrated form and the cells are then allowed to settle onto a slide.

The end result is a monolayer of cells on a slide. This method has not demonstrated any superiority over the ThinPrep pap test. It has all of the limitations of the ThinPrep pap test and also comes at an increased cost relative to the conventional pap smear test. More importantly it still does not solve the problem of having a cytotechnologist or pathologist select the abnormal specimen primarily, instead of some automated method. In today's age of technology it does not make much sense that a screening method for cervical scrape specimens require the manual examination of such a specimen under the microscope for an abnormality.

This is a very labor intensive and specific test. As mentioned earlier, review of a pap smear is the most labor intensive, least paid, and most litigious specimen that is examined under the microscope. Screening tests are designed for their high sensitivity, which usually comes at the cost of reduced specificity. That is, when designing a screening test one wants to make sure that it identifies all or as many of the true positives as possible even at the cost of having a number of false positives.

Take for instance the examination of a patient's peripheral blood. This test is first examined by a machine, which gives a complete blood count with an automated differential count of cell types in the blood.

If there is a population of cells outside the reference range or if there are a significant number of atypical cell forms identified, the machine will flag the case for review.

The pathologist would then review the peripheral smear of the patient and provide a more specific analysis of all blood cell types, shapes, numbers, and quality.

A specific differential or definitive diagnosis would then be generated. Such an automated screening test followed by either selective specific review by a pathologist or yet another automated specific test is what is required for the examination of cervical scrape specimens. Such a screening test, as with all screening tests, would need to meet certain criteria to justify its introduction into the clinical arena. Three of these are as follow: Image analysis technology has been researched in an attempt to fully automate the screening of monolayer preparations of cervical scrapes.

One such technology named Papnet still requires obligatory review of a certain number of fields selected by the contputer which displays them onto a monitor for such. Another form of automated image analysis technology called AutoPap does. Neither of these technologies has reached clinical diagnostic acceptance. A proposed alternative approach would be to perform molecular diagnostic testing for HPV as a screening methodology for all cervical scrape specimens.

According to a study published in the Journal of Pathology, HPV is present in virtually all cervical cancers, an estimated Obstet Gynecol ; Salomon D, et al. Comparison of three management strategies for patients with atypical squamous cells of undetermined significance: Baseline results from a randomized trial. J Natl Cancer Inst. Currently HPV testing can be done by either in- situ hybridization or hybrid capture methodologies to identify specific sequences of the HPV genome.

That is, a large percentage of women who are HPV positive have no identifiable lesion, nor do they ever go on to develop lesions.

These are thought to be subclinical infections. Therefore implementation of this technology as a screening protocol would give rise to oversensitive and unmeaningful results that would be too expensive. Unlike the Hybrid capture HPV DNA test, pap smears which have been on the market for several decades, are currently o fered at levels equal to production costs are not likely to become less expensive, in fact, the introduction of fluid based methods of collection while making it possible to perform HPV testing from such have drastically increased the cost of the pap smear.

The proposed method of automated cervical cytology screening would of er advantages over both current morphology and HPV testing methodologies.

The performance of both methods of detection directly on the fluid suspended cells would eliminate the cost incurred by slides, filters, and machinery used for current monolayer preparations Thinprep. In addition to the elimination of these costs, the application of automated morphology and HPV DNA testing directly from the cell suspension also offers the potential for drastic cost reduction with increased volume. In addition to the limitations described, this comes at an enormous financial burden.

There are approximately 50 million pap tests performed in the United States annually. Accordingly, a successful automated pap screening machine would eliminate human screening of 45 million pap smears per year. This device would 1 test cells directly from a fluid medium, 2 analyze all of the fluid, 3 eliminate human evaluation of normal specimens, 4 have good sensitivity, 5 have a meaningful positive and 6 be cost effective.

The specimen is collected in a fluid based medium and the suspended cells are analyzed by means of flow cyto etric technology. Direct analysis of the cells in a fluid based medium with minimal manipulation would allow for analysis of such cells in an environment closer to in vivo than any other method currently available.

This would also obviate the need to make a slide preparation of the sample to be analyzed, a prerequisite of any other currently available method. The elimination of slide preparation translates to reduction of time, morphologic artifact, and cost. In one embodiment of the invention, analyzed samples can be recollected for further morphologic visual analysis or molecular DNA analysis.

Alternately, residual unprocessed fluid can be subjected to such analysis. In another embodiment of the invention, specific atypical cell populations within a sample can be sorted and collected for further visual morphologic or molecular DNA analysis fig. Data relating to the cellular viability, number of cells examined, and relative percentage of each population type will be displayed in both a tabulated form fig.

Cell populations with significant atypia will be automatically flagged as abnormal. In yet another embodiment of the invention, flow cytometric technology will be used in conjunction with DNA fluorescent in-situ hybridization FISH technology to detect human papilloma virus HPV infected cell populations.

Identification of such populations will be correlated with morphologic flow cytometric information. All the information can be displayed simultaneously in 3 dimensional graphical form fig.

Alternately, specific atypical populations can be gated and analyzed for positivity of both high risk and low risk DNA probes fig. As before, cellular populations that are HPV DNA positive with morphologic atypia can be selectively sorted for morphologic confirmation fig. Accordingly, it is an object of the present invention to provide an improved system for cytological screening. It is a further object of the present invention to provide an improved method for cytological screening.

The subject matter of the present invention is particularly pointed out and distinctly claimed in the concluding portion of this specification.

However, both the organization and method of operation, together with further advantages and objects thereof, may best be understood by reference to the following description taken in connection with accompanying drawings wherein like reference characters refer to like elements.

Detailed Description According to a preferred embodiment of the present invention, automated systems and methods are provided. The transport medium should have several properties.

The device first agitates the specimen gently to disrupt residual cellular groups. As mentioned earlier this may also be done manually prior to the introduction of the specimen to the device. The device then suctions the fluid based specimen from the vial through negative pressure. The method of specimen analysis is through flow cytometry technology. Flow cytometry is a process by which cells pass singly in a fluid stream.

The exact methods of achieving this may vary. It may be achieved by suspending the cells in isotonic fluid medium and introducing it into a nozzle shaped chamber with a small exit diameter. Isotonic fluid is also introduced into a sheath chamber surrounding the sample chamber in order to create a laminar flow.

The differential pressure under which the sample and the sheath fluid are forced into the nozzle results in concentrating the cells in the center of the fluid stream in which the pass singly.

The second component involves the detection of the cells and the properties of such as they pass singly. The three principle features used to establish morphology are 1 nuclear size 2 nuclear surface irregularity and 3 cell size. The nuclear to cytoplasmic ratio is a 4 th - calculated parameter. This is accomplished by passing a beam of light typically a laser beam through the laminar cell flow.

Photons are separated and collected by both forward and side light scatter detectors. The forward detector is placed directly in line with the direction of the light beam on the opposite side of the laminar low or cells being analyzed. The side light scatter detector is usually placed at a 9.

Photo multiplier tubes then convert the detected light signal into a digital signal so that the data can be analyzed and finally be plotted into a dot plot graph or screen FIG. In order to differentiate between nuclear size and cytoplasmic size several methods may be employed.

This is an old technique used by old coulter counters, see, for example, U. This method can be combined with a nuclear stain. By using such a stain the degree of light transmission detected by the forward photomultiplier detector reflects the nuclear size.

In this way the differential light transmission detected in the forward photomultiplier tubes can differentiate between nuclear size and cell size. A change in the electromagnetic field caused by the passage of a cell is measured. This measures the cell size. Again this can be combined with a nuclear stain such as a DNA stain. As before the degree of transmitted light detected by the forward photomultiplier tube correlates with nuclear size. See, for example, U. Since cervical end endocervical cells have very little cytoplasmic granularity the detected light by the orthogonal photomultiplier tube will predominantly represent nuclear surface irregularity.

Traditionally the orthogonal photomultiplier tube has been used by flow cytometers to represent cytoplasmic granularity. Its use for detection of nuclear surface or perhaps internal nuclear irregularities would require calibration and correlation between morphologically normal and abnormal cells. Also, variation index between cells may be used in detecting abnormal nuclear features.

Occasionally squamous cervical epithelial cells do have so called keratohyalin cytoplsmic granules observed with a Pap stain.

Such granules are said to be a soft criteria for dysplasia. But since both nuclear surface irregularities, and the presence of keratohyalin granules are features of dysplasia their contribution to cellular complexity is complementary. A stain can be used that will stain both the nucleus and cytoplasmic granules. The criteria used to identify squamous intraepithelial lesions and glandular lesions may include but are not limited to the following: The following parameters may be employed, for example: The process involves six steps.

This procedure would require the need to permeabilize the cell membrane. The permeabilization step provides the mechanism by which the fluorochrome-conjugated probe can enter the cell so that nuclear binding by the probe can take place.

Their nucleic acid sequences molecules are however protected by cross-linked proteins. These proteins would prevent a nucleic acid probe from gaining access the cell DNA.

In order to overcome this problem the liquid based cells undergo digestion by a protean specific enzyme or protease. The double helix is held together by hydrogen bonds. The method by which the DNA hydrogen bonds are broken so that the individual strands of DNA unfold is called denaturation. This can be accomplished either by heat or chemical methods. The hydrogen bonds between the probe and the cells DNA are however weak and the probe is in constant competition with the cells complementary DNA.

In order to solidify the probes hydrogen bonds to the cells DNA the temperature is either decreased or the chemical conditions for denaturation are neutralized. These four steps can be performed in a fluid state and automated by sequentially adding the appropriate enzyme or chemical.

Each enzyme or chemical used can also be neutralized by the addition of a neutralizing agent before proceeding to the next step. These are washes that are performed after the hybridization step. The purpose of the washes is to remove any. The washes also serve to remove probe that has bound to undesired portions of DNA nonspecifically. The binding of probe to such undesired segments of DNA is less stable than the more specific areas that the probe was designed to bind.

The stringency of the washes can therefore be manipulated such that the probe is removed from the undesired nonspecific portions of DNA and not the specifically desired segments. The stringency of the washes of course requires certain research and development. Another area is the identification of a method of performing these washes in a fluid based specimen altogether. These may be intracellular or not. However the component to which the probe is bound is usually attached to a solid state.

In this way the bound probe is kept from being washed away. The cells in a fluid based specimen may have to temporarily be attached to a solid state for the washes to be effective. Otherwise a differential gradient may be devised such that the wash with unbound probe will wash ahead of the intracellular bound probe. Other alternatives may be devised.