150 results about "Formalin fixed" patented technology

The present invention provides novel methods for analyzing gene expression levels from fresh or aged (more than one year old) formalin-fixed, paraffin-embedded tissue (“FFPET”) samples that comprise pre-hybridizing a labeled nucleic acid sample prepared from the formalin-fixed, paraffin-embedded tissue sample with a first microarray, hybridizing the unbound labeled nucleic acid sample with a second microarray, and detecting the labeled nucleic acid sample bound to the second microarray. The pre-hybridization step results in an increase in the specific gene signals in subsequent hybridizations with high density gene expression arrays. The first microarray used for the pre-hybridization step can be either a new or used microarray. Importantly, from a cost-savings perspective, the inventors determined that when the first microarray used for the pre-hybridization step is a previously used microarray, the results of the subsequent hybridization on a second microarray are nearly identical to the results obtained when the pre-hybridization was carried out using a new or previously unused microarray.

The invention provides an automated method of single cell image analysis which determines cell population statistic, applicable in the field of pathology, disease or cancer diagnosis, in a greatly improved manner over manual or prior art scoring techniques. By combining the scientific advantages of computerized automation and the invented method, as well as the greatly increased speed with which population can be evaluated, the invention is a major improvement over methods currently available. The single cells are identified and displayed in an easy to read format on the computer monitor, printer output or other display means, with cell parameter such as cell size and staining distribution at a glance. These output data is an objective transformation of the subjective visible image that the pathologist or scientist relies upon for diagnosis, prognosis, or monitoring therapeutic perturbations. Using our novel proposed technology, we combine the advantages provided by the clinical standard tool of flow cytometry in quantifying single cells and also retain the advantages of microscopy in retaining the capability of visualizing the immunoreactive cells. Unlike flow cytometry however, the invention uses commonly available formalin fixed immunostained tissue and not fresh viable cells. To accomplish this aim, we resort to new and improved advanced image analysis using a unique, useful, and adaptive process as described herein. The method uses multi-stage thresholding and segmentation algorithm based on multiple color channels in RGB and HS I spaces and uses auto-thresholding on red and blue channels in RGB to get the raw working image of all cells, then refines the working image with thresholding on hue and intensity channels in HS I using an adaptive parameter epsilon in entropy mode, and further separates different groups of cells within the same class, by auto-thresholding within the working image region. The Immunohistochemistry Flow cytometry (IHCFLOW) combination results in a new paradigm that is both useful, novel, and provides objective tangible result from a complex color image of tissue.

This invention provides methods of quantitating nucleic acids from problematic samples, such as aged samples, formalin fixed samples, paraffin embedded samples, samples with aneuploid cells, and cells with fragmented nucleic acids. Methods include techniques to efficiently solublize the nucleic acids under non-denaturing conditions from preserved clinical samples without resort to organic extractions, to normalize cell counts regardless of aneuploidy, to access the fragmentation state of the nucleic acids, and to provide standard curves for degraded nucleic acid samples.

The invention provides methods for multiplex analysis of biological samples of formalin-fixed tissue samples. The invention provides for a method to achieve a multiplexed, multi-staged plurality of Liquid Tissue preparations simultaneously from a single histopathologically processed biological sample, where the protocol for each Liquid Tissue preparation imparts a distinctive set of biochemical effects on biomolecules procured from histopathologically processed biological samples and which when each of the preparations is analyzed can render additive and complementary data about the same histopathologically processed biological sample.

The present invention relates to the field of cancer. More specifically, the present invention provides methods and compositions useful for diagnosing or predicting cancer in a patient. In one embodiment, a method for identifying a patient as having cancer comprises the steps of (a) providing a formalin-fixed, paraffin-embedded or fresh frozen sample of patient tissue; (b) steaming the sample in antigen retrieval buffer; (c) incubating the sample in hydrochloric acid (HCl); (d) incubating the sample with an affinity reagent specific for 5hmC under conditions to form a complex between the affinity reagent and 5-hydroxymethylcytosine (5hmC) present in the sample; (e) detecting the complexes formed between 5hmC and the affinity reagent with secondary detection reagents; (f) quantifying 5hmC levels; and (g) identifying the patient as having cancer if the 5hmC levels in the sample are reduced as compared to a control.

The present application relates to methods for increasing stability of formalin fixed, optionally paraffin embedded biological material. In one example, DNAse inhibitors and / or RNAse inhibitors are combined with the biological material or formalin at the time of fixation or shortly prior.

Molecular characterization of disease has become the dominant trend in modern medicine, and it has recently become increasingly important to obtain qPCR, microarray, and next-generation sequencing (NGS) data for both research and clinical applications. Formalin-fixed, paraffin-embedded (“FFPE”) samples have become the standard way of storing clinical biopsies throughout the world. Unfortunately, the poor quality and quantity of nucleic acids obtained from such specimens has posed severe limitations on the types of studies that can be accomplished. Existing methods for retrieval of the biomolecules from their crosslinked matrices are poorly effective and rely on harsh conditions which further damage the biomolecules being extracted. The invention provides compositions and methods for retrieval, analysis and use of biomolecules, including nucleic acids, from such samples.

A formalin fixed paraffin block an organ is fixed on a stage, the stage is moved and a front end of a tubular blade is relatively positioned at a target part, the tubular blade punches the tissue, the tissue is extracted by a tissue extracting rod, a tissue block has been prepared in advance having a plurality of holes having inner diameter very close to the outer diameter of the tubular blade, the extracted tissues are planted in the holes on an embedding pan so that the front end of the tissue is protrusively facing toward the embedding pan, and liquid paraffin is filled into a gap between the embedding pan and the tissue block and solidified, whereby the tissue and paraffin are integrated, and good array block sections are constructed easily.

The disclosure provides methods for the use of gene expression measurements to classify or identify neuroendocrine cancer in samples obtained from a subject in a clinical setting, such as in cases of formalin fixed, paraffin embedded (FFPE) samples.

The present teachings provide methods, compositions, and kits for detecting micro RNAs (miRNAs). In some embodiments, the miRNAs are quantified from formalin-fixed paraffin-embedded tissue samples in which messenger RNA is degraded. The present teachings take advantage of the observation that most mature miRNAs in vivo are protected by degradation as a result of their association with RISC. Thus, novel methods of studying nucleic acids in archived tissues containing degraded messenger RNA are provided, wherein RISC-protected miRNAs are liberated, and analyzed.

The invention discloses a quantitative sequencing and library building method for a fusion gene on the basis of DNA (Deoxyribonucleic Acid). The quantitative sequencing and library building method comprises the following steps: firstly, constructing a genome fragmentation DNA library and purifying the library; secondly, capturing a fusion gene generation region by PCR (Polymerase Chain Reaction) amplification, purifying the captured gene and enriching a sequence containing a specific primer fragment; thirdly, capturing a target fragment containing the fusion gene by nested PCR amplification; fourthly, constructing a DNA sequencing library with high-throughput sequencing. The invention also discloses a quantitative sequencing and detecting method for the fusion gene by using the DNA sequencing library prepared by the quantitative sequencing and library building method, application of the quantitative sequencing and detecting method as well as a detection kit containing the DNA sequencing library. According to the quantitative sequencing and library building method disclosed by the invention, the downstream where a fusion breakpoint occurs is anchored by using a one-way specific primer; a target sequence is obtained by pairing specific primers with universal primers and using a PCR method; the background is further reduced label enriching and nested PCR, so that the specificity is improved, the time for building the library is shortened, and the cost for building the library is reduced; the quantitative sequencing and library building method is suitable for an FFPE (Formalin Fixed And Parafiin Embedded) sample or liquid biopsy.

A method for fixation of organic tissue samples includes immersing a tissue sample in a solution of Formalin at a temperature of between 2° C. and 10° C. for a first time period (A), and immersing the tissue sample in a cold fixation dehydrating agent for a second time period (B) following the first time period. An automated tissue fixation system for fixating organic tissue samples in Formalin is also described.

The invention discloses an FFPE reference product for gene detection and a preparation method and application of the FFPE reference product. The preparation method comprises the following steps that S1, cell culture is performed on a tumor cell line, and cell pellets are collected; S2, formalin fixation is performed on the cell pellets, then sepharose gel wraps the cell pellets to form cell aggregates, and the cell aggregate are prepared into cell paraffin blocks; S3, genomic DNA of the tumor cell line in the cell paraffin blocks is extracted, and genetic mutation frequency determination is performed on the genomic DNA of the tumor cell line; S4, the genomic DNA of the tumor cell line containing a target mutation site is mixed into a DNA mixture of a target mutation frequency, and the DNAmixture is the FFPE reference product for gene detection. According to the technical scheme, formalin fixation and paraffin wrapping are performed on the cells cultured by tumor cell line, so that thesituation of clinical samples can be better simulated.

This invention provides methods of quantitating nucleic acids from problematic samples, such as aged samples, formalin fixed samples, paraffin embedded samples, samples with aneuploid cells, and cells with fragmented nucleic acids. Methods include techniques to efficiently solublize the nucleic acids under non-denaturing conditions from preserved clinical samples without resort to organic extractions, to normalize cell counts regardless of aneuploidy, to access the fragmentation state of the nucleic acids, and to provide standard curves for degraded nucleic acid samples.

The invention discloses a specimen preserving, fixing and accelerating agent which comprises the following components in percentage by weight: 2-20% of main additive, 0.2-5% of auxiliary additive and the balance of ethanol solution of which the concentration is 50vt%, wherein the main additive is selected from laurocapram, thiazone, menthol or isopropyl myristate; and the auxiliary additive is selected from disodium ethylene diamine tetraacetate, ethylene diamine tetraacetic acid tetrasodium salt, ethylene diamine tetraacetic acid dipotassium salt, ethylene diamine tetraacetic acid tripotassium salt, ethylene diamine tetraacetic acid calcium disodium salt or ethylene diamine tetraacetic acid zinc disodium salt. The specimen preserving, fixing and accelerating agent can effectively improvethe velocity of permeability of an environment-friendly non-formalin fixing solution in human bodies, animals and plant tissues, shorten the fixing time, and improve the efficiency and the quality for manufacturing pathological sections by a pathology department.

The invention relates to a method for extracting desoxyribonucleic acid from formalin fixed and paraffin embedded tissues, and belongs to the technical field of nucleic acid application in biology. The method comprises the following steps of: preparing special lysis solution, adding the lysis solution into paraffin section tissues, boiling at high temperature for 30 minutes, and centrifuging to take supernate; adding absolute ethanol for uniform mixing, and adding the mixed solution into a silicon membrane absorption column for centrifuging; rinsing by using protein-free liquid and salt-free rinsing liquid; and eluting by using elution buffer. In the method, a toxic reagent of dimethylbenzene is not used for dewaxing, and harm to bodies of experimenters is avoided; and precious protease Kis not needed to perform long-time incubation enzymolysis, the operation is simple and quick, the extracted desoxyribonucleic acid in genome is high in quality and stability, and the cost and time can be saved to the greatest degree. The extracted product is subjected to polymerase chain reaction (PCR) detection, long segments with about 750pb can be obtained through amplification, and the work in the aspects such as scientific research, biomedicine and the like is greatly facilitated.

The invention relates to a buffer system comprising maleic acid and maleic acid dipotassium salt, for use with formalin-containing fixative solutions. The buffer system is soluble / miscible in dehydration solutions including alcohols, ketones, and monomethyl ethers, thereby preventing formation of precipitates in fluid lines and valve systems of automated tissue processors.

Ligation-enhanced nucleic acid detection assay embodiments for detection of RNA or DNA are described. The assay embodiments rely on ligation of chimeric oligonucleotide probes to generate a template for amplification and detection. The assay embodiments are substantially independent of the fidelity of a polymerase for copying compromised nucleic acid. Very little background amplification is observed and as few as 1000 copies of target nucleic acid can be detected. Method embodiments are particularly adept for detection of RNA from compromised samples such as formalin-fixed and paraffin-embedded samples. Heavily degraded and cross-linked nucleic acids of compromised samples, in which classic quantitative real time PCR assays typically fail to adequately amplify signal, can be reliably detected and quantified.

Methods of reliably quantifying telomere length in cells or tissues that have been formalin fixed and paraffin embedded (FFPE) samples by quantitative polymerase chain reaction protocol and kits for use with such various methods are provided. The methods of the present invention may be used to predetermine an individual's response to treatment with a telomerase inhibitor, a telomere damaging agent or a telomerase activator.

The invention relates to a device for detecting copy number variation of FFPE (formalin-fixed and paraffin-embedded) samples. The device for detecting the copy number variation of the FFPE samples comprises a sequencing data acquisition module, a sequence comparison module, an earlier-stage data processing module, a normalization module, a background library screening module, a data fluctuation elimination module, a GC (guanine and cytosine) correction module and an output module. The device has the advantage of high detection sensitivity.

The invention discloses a method for producing a double-staining plastic-sealed specimen of fish bones. The method comprises the following conditions and steps: cleaning internal organs, scales and skin of fish bodies of fishes with hard and soft bones, fixing by virtue of 10% formalin, soaking with distilled water for 1-2 days so as to completely remove formalin, staining skeletons and soft bones blue by virtue of a dye, namely alcian blue, processing by virtue of alcohols of different concentrations, processing muscles of the fish bodies by virtue of trypsase until the muscles are in a transparent state, simultaneously maintaining the blue of the soft bones, staining the hard bones into red by virtue of alizarin red, carrying out re-transparentization by virtue of different concentrations of mixed solutions of 0.5%KOH and glycerin, plasticizing the fish bodies by virtue of polyvinylpyrrolidone, after the plasticization, carrying out air drying on the specimen, and finally sealing the specimen by virtue of epoxy resin. According to the finished specimen, the skeleton specimen including red hard bones and blue soft bones can be directly seen through the resin and the plasticized muscles.

The invention provides a method for extracting DNA from formalin-fixed tissue. The method combines a formalin removal method with a conventional DNA isolation method, can extract high-quality genomic DNA from the formalin-fixed tissue, and has the characteristics that: the method has low cost and is easy to popularize and apply; and the obtained DNA is applicable for PCR amplification, and the like.

The invention provides a wax cell chip inoculation method which is characterized by comprises the following specific steps of: wrapping cells by using casings which are subjected to fixation by formalin, treating and dipping wax so as to obtain wax-dipped cell specimens; heating to the wax-dipped cell specimens to be in a molten state, opening the casings, scrapping the cells and liquid wax into an EP (Epoxy Resin) tube, uniformly mixing, molding into the cell chips in desired shapes, putting a wax mold preparation frame on a wax mold plate, pouring the wax into the wax mold preparation frame, polymerizing, taking gout and detaching down the wax mold plate; putting an obtained wax mold into an incubator together with the wax mold frame, slightly softening the wax mold and taking the softened the wax mold out, drilling holes within an inoculation range of the wax mold, and inoculating the cell chips into corresponding holes of the wax mold; putting the wax mold into a constant temperature incubator to be polymerized; and putting the wax mold and the wax mold frame into a refrigerator, taking the wax mold and the wax mold frame out, separating the wax mold from the wax mold preparation frame by laterally knocking the wax mold preparation frame, and slicing the obtained wax mold so as to obtain the wax cell chips. The wax cell chip inoculation method has the advantages of low cost, convenience, rapidness, high yield and diversity in molding shapes.

The present invention relates to the field of cancer. More specifically, the present invention provides methods and compositions useful for diagnosing or predicting cancer in a patient. In one embodiment, a method for identifying a patient as having cancer comprises the steps of (a) providing a formalin-fixed, paraffin-embedded or fresh frozen sample of patient tissue; (b) steaming the sample in antigen retrieval buffer; (c) incubating the sample in hydrochloric acid (HCl); (d) incubating the sample with an affinity reagent specific for 5hmC under conditions to form a complex between the affinity reagent and 5-hydroxymethylcytosine (5hmC) present in the sample; (e) detecting the complexes formed between 5hmC and the affinity reagent with secondary detection reagents; (f) quantifying 5hmC levels; and (g) identifying the patient as having cancer if the 5hmC levels in the sample are reduced as compared to a control.

An ultra-sensitive, specific methodology for detecting PIK3CA mutations in biological samples of cancer patients, comprises a combination of allele-specific, asymmetric rapid PCR and melting analysis in a DNA sample from Circulating Tumor Cells, cell-free DNA in plasma / serum, or Formalin-Fixed Paraffin-Embedded tissues. Using the allele-specific primers for hotspot mutations in exons 9 and 20 (E545K and H1047R), detection can enhance amplification of mutant PIK3CA allele sequence, whereas presence of corresponding competitive blocking unlabeled probes for each exon can avoid non-specific amplification of wild-type PIK3CA sequence increasing the sensitivity and the specificity of method. The mutational detection is completed with melting curve analysis of the unlabeled probe and DNA template of the mutant PIK3CA sequence. Evaluation of PIK3CA mutational status on CTC in peripheral blood and cfDNA in plasma / serum of patients has potential for clinical applications and therapeutic interventions, since presence of PIK3CA mutations is associated with response to molecular targeted therapies.

The invention discloses an automatic extraction method for nucleic acid in an FFPE (Formalin-Fixed and Parrffin-Embedded) sample. When DNA or RNA of FFPE is independently extracted, cell tissue can beconfined in a certain space range by magnetic beads through a magnetic separation device, the consequence that tissue is sucked off along with a dewaxing agent or other solution can be effectively avoided, and furthermore the DNA or RNA of FFPE can be extracted; in co-extraction of the DNA or RNA of FFPE, the tissue is dewaxed and rehydrated along with the magnetic beads, lysate RTL is added, andthen RNA can be released into the solution. A centrifugal tube is placed in the magnetic separation device, digestion lysate with the RNA is transferred into a new centrifugal tube by using a liquidtransferring device, and then RNA extraction can be carried out; lysate DTL is continuously put into the magnetic beads into which the cell tissue is adsorbed in the original centrifugal tube, then the DNA can be released into the solution, and DNA extraction can be carried out.

The invention discloses a method of extracting DNA from formalin-fixed hair stems. After washing and pretreatment, the hair stems are put into a homogenizer full of 0.01mmol / L sodium citrate solution for grinding under 0 to 10 DEG C, then put in boiling water bath for 5 to 15min and be restored; ethanol is added for stewing, then eccentrically collected and deposited; hair digestive fluid and proteinase K solution are added into the deposition, and continuously digested for 1 to 2 hours in water bath at the temperature of 37-57 DEG C; DNA is extracted after digestion. The invention restores hair stem samples so as to totally or partially recover the activity of the protein active site. In addition, the preparation of the digestive fluid takes the characteristics of hair stem crack into full consideration so as to greatly improve the efficiency of hair stem crack and shorten the time of hair digestion. The method is used to extract not only the genome in the hair stem soaked in formalin but also the genome in the ancient hair stem.

The invention discloses a making method for a skeleton-dyed specimen plastic package specimen of cartilaginous fish. The making method comprises the following conditions and steps: cleaning internal organs, scales and the skin of a fish body, fixing the fish body with 10 percent of formalin, soaking the fish body with distilled water for 1 to 2 days to make sure that the formalin is completely cleared away, dyeing the skeleton and cartilages into blue with a dye alcian blue, performing treatment with alcohol at different concentrations, then treating muscles of the fish body with trypsin till a transparent state is achieved, retaining the blue color in the cartilages, dyeing hard bones into red with alizarin red, performing transparent treatment again with 0.5-percent KOH and glycerinum mixed solution at different ratios, then plasticizing the fish body with polyvinylpyrrolidone, after the fish body is plasticized, drying the specimen in air, and finally sealing the specimen with epoxy resin. According to the completed specimen, the skeleton specimen in which the hard bones are red and the cartilages are blue can be seen directly through the resin and the plasticized muscles.

The present invention discloses a method for rapidly concentrating and extracting nucleic acid target cells from a sample, wherein the method comprises the steps of fixing surgical or biopsy specimens with neutral formalin, dehydrating to be embedded in paraffin, cutting multiple white slices and selecting one for HE staining, observing tumor tissue cell distribution of HE stained slices under an optical microscope, and marking the distribution area of target cells; baking the remained white slices for xylene dewaxing, scraping the tissue rich in target cells on the white slices according to the marked HE stained slices for target cells concentrating, and extracting nucleic acid from the concentrated target cells. The method can improve concentration of nucleic acid in the target cells to up to more than 40% to 60% so as to raise positive rate of molecular pathology detection and reduce false negative rate without any expensive equipment or specialized complex technology with almost no obvious time consumption, therefore the method is suitable for being applied in all inspection department of pathology and biological laboratories and is used to provide more accurate molecular diagnosis for clinical diagnosis and treatment.