WO2012078138A1 - Dispositif de contrôle qualité d'une matrice de cellules pour analyse pathologique - Google Patents

Dispositif de contrôle qualité d'une matrice de cellules pour analyse pathologique Download PDF

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Publication number
WO2012078138A1
WO2012078138A1 PCT/US2010/059205 US2010059205W WO2012078138A1 WO 2012078138 A1 WO2012078138 A1 WO 2012078138A1 US 2010059205 W US2010059205 W US 2010059205W WO 2012078138 A1 WO2012078138 A1 WO 2012078138A1
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Prior art keywords
assay
quality control
array
cytokeratin
panel
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PCT/US2010/059205
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English (en)
Inventor
Wei-Sing Chu
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Wei-Sing Chu
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Application filed by Wei-Sing Chu filed Critical Wei-Sing Chu
Priority to PCT/US2010/059205 priority Critical patent/WO2012078138A1/fr
Priority to CN201080034906.5A priority patent/CN103347574B/zh
Publication of WO2012078138A1 publication Critical patent/WO2012078138A1/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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers

Definitions

  • the present invention relates generally to a method of making and using quaiity control siides upon which cultured ceils are placed in a Sow density array format. Since the properties of the cultured cells on the slides are known or can be clearly known by studies, they can be used as positive and negative controls for the process of histopathoSogica! analysis such as
  • ceil array quality control slides can replace tissue-based quality control siides w ich are often hard to come by,
  • Pathological analysis such as IHC, ICC, and SSH, are routineiy conducted on patient specimens for diagnostics and prognostics for cancer and other diseases. Such analysis often includes complex steps and must be carefully controlled.
  • a typical IHC, ICC, or !SH assay requires a series of many steps. These include: obtaining a biological sample such as a biopsy, fixing the sample in formalin, processing the sample by dehydrating and clearing, embedding the sample in paraffin, cutting serial sections and mounting on microscope slides and drying. These steps are followed by steps to deparaffimze
  • hematoxylin and eosin stain for example, nuclei are colored purple (with hematoxylin) and the cytoplasm red/pink (with eosin). Often, the type of information available from such stains is insufficient for accurate diagnosis. !HC and ISH stains have the ability to extend the level of analysis on a biopsy sample, beyond eeilular size and shape, to a molecular level. Tissue samples can be probed for the presence of specific proteins and genes using antibodies and nucleotide probes.
  • the presence or absence of positive signals of certain assay targets or biomarkers can be indicative of the ce!iuSar iineage or genomic mutations of a tumor, facilitating a diagnosis or prognosis with certain types of anti-tumor therapies.
  • the presence of microbiologic agents, such as viruses or bacteria can be detected using appropriate antibodies or nucleotide probes.
  • tissue samples previously documented to contain the particular antigen as positive controls.
  • tissue controls are depleted, new tissues/tumor samples are procured to replace those expended.
  • each antibody is tested on a positive tissue control. In this manner, each antibody is validated at a specified dilution.
  • the improvement involved embedding tissue cores in a paraffin block.
  • the cores were cut from the tissue of origin with the use of an ordinary plastic drinking straw.
  • tissue block controls provides a big burden for histology technicians in collecting excess tissue samples, preparing control slides, and characterizing their antigenic properties.
  • Many researchers tried using various non-tissue materials such as cultured cells and even peptides.
  • Cultured cells with known staining properties spotted on glass slides are often used by researchers and commercial providers of IHC diagnostic kits to act as positive and negative controls (A. Rhodes, 2002, Am. J. Clin. Pathol. P81-89).
  • MoskaSuk et al C, A. Moskaluk and , H.
  • the present invention relates to a wide-spectrum quality control device based on cell arrays and methods for making and using such a device for performing quality control in
  • One aspect of the invention is a quality control cell array that comprises discrete dots of cultured cells derived from a variety of tumor or cancer types including carcinoma, melanoma, sarcoma, leukemia/lymphoma, and tumors of the neural system.
  • the cultured cells of one type or a mixture of cultured ceils of two to three different types are laid on the surface of a solid substrate in a distinct area, referred to as cell dot.
  • the cell array can be spotted on a microscopic slide or on a film or membrane that can be attached to a microscopic slide.
  • the quality control ceil array must include lines of cells derived from: carcinomas and iymphomas. Another embodiment of the invention is that the ceil array must further include lines of cells derived from melanomas, sarcomas, or tumors of the neura! system. Another embodiment is that each line of va!s included in the quality control cell array serve as both positive control for some assays and negative control for other assays. Another embodiment of the invention is that ceils included in the quality control cell array are cultured in vitro and treated with chemicals such as formaldehyde, g!uraldehyde, alcohol, acetone, acetic acid, xylene, or xylene substitute, wax, and paraffin.
  • chemicals such as formaldehyde, g!uraldehyde, alcohol, acetone, acetic acid, xylene, or xylene substitute, wax, and paraffin.
  • the quality control cell array contains less than 50 ceil dots. Another embodiment is that number of ceils included in a eel! dot is on the order of 10 5 to 10 4 Another embodiment is that each discrete eel! dot includes either a pure population of cultured ceils or a mix population of different Sines of ceSSs, Another embodiment of the invention is that the quality control DCi array further comprises eels infected by microorganisms, Another embodiment of the invention is that the quality control cell array is spotted on a microscopic glass slide which comprises a label region in addition to a test region where the quality control cell array is attached.
  • the quality control eel array is spotted on a microscopic glass slide which comprises a label region and a sample region in addition to a region where the quality control ceil array is attached.
  • a label to be attached on the label region comprising a patient or sampie identifying indicia, an assay target ⁇ or antibody/probe) identifying indicia, a pre-determined expression pattern of the assa target on the quality control ceil array printed on the label, and optionally a date of the assay.
  • the quality control ceil array device further comprises identification of a panel of assay targets detected by assay procedures to be controlled.
  • the quality control cell array device further comprises a predetermined dataset that relates the panel of assay targets with the quality control ceil array.
  • Another aspect of the invention is a method of making a quality control cell array device.
  • a pane! of assay targets is determined for the assay procedures in which qualities are controlled by the quality control cell array device.
  • a plurality of ceil lines are determined to be included in the quality control cell array in such a way that each assay target is expressed at a positively detectable level I at least one selected Sine of cells.
  • Assay targets refer to proteins, genes, and RNA molecules present or absent in test samples as well as cells included in a quality control ceil array to be qualitatively or quantitatively detected in an assay procedure.
  • An assay procedure in this invention refers to immunohistochemistry, immunocytochemistry, and in situ hybridization, !n the case of immunohistochemistry or immunocytochemistry assay, the assay targets are protein antigens to be detected in test samples.
  • the assay targets refer to DMA or RNA sequences,
  • One embodiment of the invention is that the pane!
  • assay targets pencytokeratms, EMA, $-100, HMB45, smooth muscle specific antigen, E , PR, Her2, P53, PSA, vimentin, ehromogransn, desmin, CDS, CD2Q, CD117/c-ktt, CD15, CD30, CD45, CD99, Bcl-1 ⁇ cyclin Dl), Bcl-2, KI-67, Ig kappa, fg lamda, and CEA.
  • pencytokeratms EMA, $-100, HMB45, smooth muscle specific antigen, E , PR, Her2, P53, PSA, vimentin, ehromogransn, desmin, CDS, CD2Q, CD117/c-ktt, CD15, CD30, CD45, CD99, Bcl-1 ⁇ cyclin Dl), Bcl-2, KI-67, Ig kappa, fg lamda, and CEA.
  • the above 26 cellular proteins
  • One aspect of the present invention is a method of using the quality control cell array device. It includes establishing a pre-determined dataset for the ceil array and related panel of assay targets; presenting said dataset on paper media or digital media; conducting an assay procedure to be controlled on a test sample and on a quality control ceil array simultaneously with test reagents for an assay target included in the related panel of assay targets; recording the test results by viewing, imaging, or digital scanning; and comparing the test signal to data in the pre-determined database.
  • One embodiment of the invention is that a pre-determined dataset is established for the quality control DC! array for an assay procedure by a process comprising the following steps:
  • the predetermined dataset for the quality control DC! array is stored in a digital media such as a CD or DVD. in this case, data can be view on a computer monitor or printed out on paper media.
  • the pre-determined dataset for the quaiity control cell array is presented on paper media by a table, a chart, or a collection of pictures.
  • Embodiments of the invention include conducting an assay procedure to be controlled on a test sample and a quality control cell array device with test reagents for detecting a specific target included in the related panel of assay targets, recording the test results generated on the quality control cell array by viewing by human eyes or imaging by a camera or a digital scanner, preferably through a microscope, comparing the test results generated on the quality control cell array with pre-determined data for the specific target included in the pre-determined dataset, and determining if a there is a match for the results generated on the quality control cell array with the pre-determined data in the pre-determined dataset.
  • Figure 1 illustrates an example of a quality control cell array device.
  • the quaiity control cell array is spotted on a microscopic test slide 1 which includes a sample region 2, a controS region 3, and a label region ( Figure la).
  • Figure lb indicates a printed label to be attached to the label region on the microscopic slide.
  • the printed label 5 includes sample !D 6, !D of the specific assay target 7, a pre-determined expression pattern of the specific assay target on the quality control cell array 8, and optionally a date of performance of the assay 9.
  • Figure lc illustrates signal indicators for test results on the quaiity control cell array.
  • Figure 2 illustrates nine ⁇ 9 ⁇ possible locations of a quality control: ceil array on microscopic slides.
  • Figure 3 is an example of a quality controi cell array. Ten cell dots are laid out in two rows.
  • the cell array includes five carcinoma cell lines, two lymphoma ceil Sines, one melanoma cell line, one sarcoma cell iine, and one CMS tumor ceil Sine.
  • Figure 4 is a schematic presentation for establishment of a predetermined dataset for the quality control ceil array.
  • An IHC or SSH assay is performed on the quality control ceil array with an antibody or probe against a specific target included in the pane! of assay targets to be controlled by the quality control ce!! array.
  • the assay results are recorded by reading the microscopic siide which carries the quality control cell array by human eyes or by a machJne ⁇ s) and stored on paper or digital media.
  • Quantitative or semi-quantitative indicators are assigned to test signals in the test results. This process is repeated for every assay target included in the pane! of assay targets in order to establish a dataset for the quality control ceil array and the related panel of assay targets.
  • Figure 5 shows a procedure for using the quality control cell array in IHC, iCC or ISH assays.
  • An I HC, ICC, or ISH assay is performed simultaneously on a test sample and on the quality control cell array under identical conditions with an antibody or probe against a target of interest included in the panel of assay targets to be controlled by the quality control cell array.
  • the assay result i.e. the signal pattern, generated on the quaiity control cell array are viewed and compared with information in the pre-determined dataset A match indicates the test on the sample is successful And a no-match indicates that the test on the sampie is failed.
  • Figure 6 shows a procedure for establishing and using a computer software and database related with the quality control cell array and the panel of assay targets.
  • the first information flow is manually typing in IDs of the assay target or Ab/probe into computer software.
  • the second information flow mediated by machines is to acquire image of stained cell array and entering target or Ab/probe ID by readi ng digital indicia (e.g., barcode) by a machine(s).
  • the computer software assigns an ID (e.g. number or barcode) for each testing Ab/probe which is associated with the staining pattern of that specific Ab/probe on the cell array in the pre-determined database stored in the computer and runs through the analytical algorism software.
  • the computer prints a staining pattern for that specific target or Ab/probe on a labei fo the manual procedure which was used by the operating personnel to compare with the staining pattern in the real assay, in the machine flow, the acquired staining image is compared with the pattern in the pre-determined dataset by the analytic algorithm and the validation results will be generated.
  • the slide reading machine can be installed in the autostamers to achieve automatic quality control.
  • the slide reading machine can also be used individually as an automatic quality control reader.
  • the present invention relates to quality control devices for immunological as well as molecular pathology and cytology analysis, and methods of using the devices, to maintain quality control of assays that measure ana!ytes in cells or tissue sections, specifically immunohistochemica! analyses of biological samples.
  • the present invention relates to methods of making quality control devices involving cell arrays and for using such cell arrays for performing quality control in related assays.
  • the present invention relates to a quality control device comprising a plurality of discrete areas of multiple types of cultured cells that ar affixed, or attached, to a reagent surface of a planar solid support.
  • the planar solid support has a front ⁇ also referred to herein as top) surface and a back ⁇ also referred to herein as bottom) surface.
  • the planar solid su port can be either a flat test platform (e.g., surface of a glass microscopic slide) or a membrane that can be adhered to the test platform by an adhesive on the bottom surface.
  • One aspect of the invention is a quality control DCi array that comprises discrete areas (dots) of cultured celis derived from tumor categories (types) including carcinoma, melanoma, sarcoma, lymphoma/leukemia, and neural system tumor.
  • the cultured celis of one type or a mixture of cultured cells of two to three different types are laid on the surface of a solid substrate in a distinct area, referred to herein as cell dots.
  • cultured viis, cancers, tissues, protein, targets, anaiytes, and samples in this application refer to those of human origin,
  • carcinoma is a tumor type derived from epithelial cells. This is the most common cancer type and represents about 80 - 90% of all cancer cases reported.
  • Sarcoma is a tumor type derived from muscle, bone, cartilage, fat or connective tissues.
  • Leukemia is a cancer type derived from white blood cells or their precursors.
  • Lymphoma is a cancer type of bone marrow derived ceils that affects the lymphatic system. Both leukemia and lymphoma are related to hematopoietic malignancy. In this application, leukemia and lymphoma are together referred to as hematopoietic malignancy. Melanoma is a malignant tumor type that originates in melanocytes, the ceils which produce the pigment melanin that colors skin, hair, and eyes.
  • Figure la is a microscopic glass slide 1 including a plurality of discrete (and distinct) cell dots 3, each consisting of a monolayer or close to a monolayer of cultured celis, in the control region, and wherein cells of one specific type or two to three different types are confined to one dots.
  • cells are covalent!y attached to the top surface of the support.
  • Cells can also be attached to the top surface of the substrate by electronic charge as in the case of attachment of tissue sections to charged glass slides.
  • the slide can also include a sample region 2 and a labeling region 4.
  • Another embodiment of the quality control device is a slide label ( Figure lb) which includes a sample identifying indicia 6, a target (or antibody/probe) identifying indicia 7, a pre-determined signal pattern 8 of the target on the cell array printed on the label, as revealed by the assay procedure to be controlled by the quality control cell array, and optionally an area for date of the assay 9,
  • Another embodiment of the quality control device is a set of schematic indicators for the predetermined signal pattern 8 to be presented ( Figure lc).
  • the schematic indicators include, but not limited to the following three sets: (A) solid dots of different fill intensities, as exemplified in Figure IcA, the lowest intensity dots represent background staining (no or undetectable expression of target/analyte), and dots of escalating levels of fill intensities each representing borderline expression (+/H low positivity ⁇ +), median positivity (++), and high positivity ⁇ +++); (8) circles with a letter inside indicating subcellular locations of assay target, as exemplified in Figure IcB, the empty circle indicating background signal (negative), the circle with a letter c indicating cytoplasm signal, the circle with a letter n indicating nuclear signal, the circle with a letter m indicating membrane signal; (C) circles with varying solid sections, as exemplified in Figure IcC, the circle with dotted line indicating background signal (negative), the black solid circle with a small white dot inside indicating cytoplasm signal, the white circle with a black dot inside indicating nuclear signal
  • Another embodiment of the quality control device is a membrane where a plurality of discrete cell dots are attached on the top surface of the membrane, and where the bottom surface of the membrane can be attached to the test surface of a planner test platform, e.g., a glass microscopic slide,
  • the ceils may be attached on the support surface as a monolayer, which is preferred, and may be as multiple layers because of cell clumping.
  • Cultured cells may be laid on th surface of solid support before fixation and processing. In this way, cells need to be fixed and processed on the solid support.
  • the preferred approach is to fix the cells by chemicals befor spotting them on the surface of solid support.
  • the processing steps generally include dehydration with alcohol, clearing with xylene or xylene substitutes, and impregnation/embedding with paraffin. These processing steps mimic the processing procedure used in preparing tissue specimens.
  • the processing steps used in preparing cell arrays in this invention can be simplified to omit dehydration and clearing steps, in this way, inert materials such as paraffin can be directly applied to ceils attached on surface of solid support.
  • Another embodiment is that cells of multipie types are embedded in paraffin to form multi-ceil- type paraffin blocks.
  • the cell blocks can then be sectioned and sections of cells are then attached to the top surface of the solid support.
  • One approach to make mu!ti-ceSi-type paraffin blocks is to bundle cylinder cores of fixed and processed cells together and embed them with paraff in.
  • Cells of a specific type can also be fixed and processed in a capillary tube that can be cut by a microtone, Multiple numbers of such capillary tubes can be bundled together and embedded by paraffin.
  • the quality control cell array can be placed on surface of a solid support together with the sample or can be placed on a separate support.
  • the support may be a microscopic giass slide and may also be a transparent film, a membrane or a slide coversiip to which quality controls and/or test samples can be attached.
  • a transparent film or membrane can be made of a variety of polymers, such as nylon, polystyrenes, polypropyienes,
  • a film or membrane refers to a solid planar subtract with a thickness of 0.001 mm to 0,5 mm.
  • the quality control cell array in present invention is a low density array which contains less than SO discrete cell dots, and preferably less than 20 cell dots, and more preferably less than 12.
  • the reason to include a low number of cell dots in the cell array of the present invention is to facilitate manufacture of the cell array device as well as observation and analysis of test results.
  • One embodiment of the invention is that the number of cetis in each DCi dot is between 10 to 10 s , and preferably 1Q 1 to 10 4 .
  • the optima! scenario is that the ceil array wiil include as Sow as possible number of celt dots and at the same time it will be able to control as high as possible number of clinically relevant assays.
  • the term assay refers to a test to detect an individual ana!yte, target, or biomarker.
  • an IHC test using an antibody to one protein target is an assay while an IHC test using an antibody to another protein target is another assay,
  • a cell line refers to a pure population of cultured cells derived from a single origin. For decades, a iarge number of ceil lines have been derived from disease tissues of almost all cancer types and origins. There are about 1000 celt lines derived from human cancers listed in the ATCC catalog. Most carroti lines are derived from tumor or cancer origins by passage or viral
  • Cultured cells contain thousands of different protein antigens and other biomo!ecules.
  • Biomarkers are present in many cultured cells in similar way as they are present in cells in disease tissue samples. Cell tines that are potentially useful in the invention are derived from various cancer types and thus the presence and absence of clinicaily relevant biomarkers are similar to those cancer cells in the cancer tissue.
  • One embodiment of the invention is that a group of highly useful cilnica! biomarkers are included in the pane! of assay targets to be controlled by the quality control cell array. A group of 26 highly useful c!inica!
  • biomarkers has been identified, including pen-cytokeratins, EMA, S- 100, HM845, smooth muscle specific antigen, estrogen receptor ⁇ ER ⁇ , progesterone receptor (PR), Her2, P53, prostate specific antigen (PSA), vimentin, chromogranin, desmin, CDS, CD20, CD117/c-kit, CD15, CD3G, CD45, CD99, Bc!-1 (cyciin 01), Bct-2, Ki-67, !g kappa, ig iamda, and CEA.
  • the above 26 cellular proteins are among the most clinical useful markers in daily immunohistochemistry or
  • immunocytochemistry tests They constitute the core pane! of assay targets to be controlled by the quality contra! cell array.
  • pane! of assay targets includes the following expanded panei of cellular proteins: cytokeratin (H W), cytokeratin (L ), cytokeratin 7, cytokeratin 20, EMA, 5-100, HM845, ART-1, tyrosinase, FLAP, hepatocyte specific Ag, muscle specific actin, smooth muscle specific antigen, ER, PR, Her2, P53, PSA, PSAP, myogenin, chromogranin, synaptophysin, desmin, vimentin, CD99, CDS, CD7, CD10, CD4, CDS, CD20, CD117/c-kit, CDS, CD15, CD 21, CD23, CD30, CD45, Bci-1 (cyciin Dl), Bcl-2, Ki-67, Ig kappa, Ig Iamda, terminal deoxynucleotidyl transferase (TdT), ca!retinin, WT-1, TTF-1
  • cytokeratin HMW
  • cytokeratin l!V W
  • cytokeratin 5 cytokeratin 6
  • cytokeratin 7 cytokeratin 8
  • cytokeratin 14 cytokeratin 18
  • cytokeratin 19 cytokeratin 20
  • EMA inhtbin, maglobtn, Ki- 67, P27, P53, terminal deoxynucieotidy!
  • TdT tartrate resistant acid phosphatase
  • TRACP 5b ⁇ tartrate resistant acid phosphatase
  • PLAP AFP, hCG, estrogen receptor (ER), Her2, EGFR, progesterone receptor ⁇ PR]
  • Zap-70, P63 prostate specific antigen ⁇ PSA ⁇
  • PSAP prostate specific acid phosphatase
  • hepatocyte specific Ag chromogranin, synaptophysin, CDla, CD3, CD4, CDS, CD7, CDS, CD 10, CD15, CD20, CD21, CD23, CD30, CD34, CD35, CD43, CD56, CD57, CD79a, CD99, CD117/c-kit, Bell fcyc!ln Dl
  • Bcl2, Bci-6 anaplastic lymphoma kinase-1 (AIk-1), fascin, Ig Kappa, ig Lamda, Pax-5, P 504s, fvlART-1, HM845, S-100, PAP
  • an expanded pane! includes the following cellular proteins and biomarkers: smooth muscle actin, muscle specific actin, Alk-1, Bel-1, Bci-2, Bd-6, BF-1, CDla, CDS, CD4, CDS, CD7, CDS, CD10, C015, CD2G, CD21, CD22, C023, CD25, CD30, CD31, C034, CD35, CD38, CD43, CD44, CD45, CD56, CD57, CD6S, CD79a, CDm/C-kit, CD138, CEA, chromogranin, c!usterin, cytokeratin (HMW), cytokeratin (LI IW), cytokeratin 5, cytokeratin 6, cytokeratin 7, cytokeratin 8, cytokeratin 14, cytokeratin 18, cytokeratin 19, cytokeratin 20, desmin, E A, fascin, granzyme B, HB E, hemoglobin, IgA, IgD, IgE,
  • microphthalmia MUC-1, MUC-2, iaminin, myogenin, HER2, EGFR, MOC-31, CD99, TAG-72, MIH ⁇ 1, M5H-2, AR, villin, melanoma assoc Ag, ACT, aipha-l-antitrysin, P27, P5G4s, inhibln, PSA, AFP, MUC-5, MUC-6, P21, flbronectin, P53, beta-catenin, CD123, CD24, CD133, adenovirus antigens, cytomegalovirus (OvIV) antigens, Epstein-Barr virus (EBV) antigens, Heiicobactor py ri antigens, human papillomaviruses ⁇ HPV ⁇ antigens, HBcAg, HSV i&IS antigens, Her2 gene amplification, c-myc gene translocation, poly-A mRNA,
  • the cell array exemplified in Figure 3 include 5 dots for carcinoma cell lines, 2 dots for lymphoma cell lines, 1 dot for melanoma eel! lines, 1 dot for sarcoma cell lines, and 1 dot for neural cancer-derived cell lines.
  • nucleic acid targets can also be controlled by the quality control cell array in ISH Assays.
  • ISH assay targets are c-myc translocation,
  • a pane! of assay targets in the iHC assay represents the protein antigens detected by antibodies used in the IHC assay procedure.
  • the term panel of assay targets can also be referred to as the panel of assays.
  • a panel of assay targets can comprise all of the above markers and even more as new clinically useful btomarkers are discovered and introduced. In this case, relatively larger number of cultured cell lines must be included in the quality control cell array, A narrowed panel of assay targets may be compiled to be suitable for specific diagnosis, prognosis as well as research needs.
  • every member in the panel must be expressed at a detectable level in at least one eel! line included in the ceil array for positive control. Every member in the panel wil! be expressed at an undetectable level in at least one other ceil Sine included in the ceil array for negative control.
  • a universally expressed assay target or marker is included in the pane! of assay targets, there will be no negative control for this specific target.
  • Universally expressed markers such as PCNA, GAPDH, beta-actin and many other cellular housekeeping proteins are expressed in ail cell Sines.
  • a nonhuman cell line maybe included in the cell assay to function as a negative control for universally expressed human biomarkers.
  • One embodiment of the invention for selection of ceil lines to be included is that for a specific non-universal assay target, !ess than 75%, 50%, 25%, or 10% of the cell dots in the quality control celi array generate positive detection signal.
  • One embodiment of the invention is that universally expressed target are specified and distinguished from other targets in the panel.
  • the term detectable level refers to strength of resulting signals generated in an assay to revea! expression or existence of a target biomarker.
  • the signals can be co!orimetric or fluorescent.
  • a detectable level refers to a signal strength that stands above the background noise.
  • An undetectable level refers to a signal strength that is undistinguishable from the background noise.
  • Proteins and nuclear acid sequences from microorganisms such as viruses, bacteria, yeast, and fungi can be included in the pane! of assay targets, If these targets are included, ceils infected or transformed by the related microorganisms are included in the quality control ceil array, 3. Method of using the cell array quality control device
  • ceil array control slides can be used alone. Or, the ceil array can be spotted in a test region on a slide that also contains a test sample on the sample region ( Figure la ⁇ . Encompassed is a method for validating, or verifying, the proper performance of an !HC, ICC, or ISH assay for the detection of the presence or absence, as well as cellular localization, of target molecules in a biological sample.
  • the method comprises simultaneously processing the biological sample and a celi array quality control device described herein in the assay to detect the presence or absence of one, or more target molecules.
  • processing means performing all the steps of an assay required to detect the presence or absence and subcellular localization, of the target molecule.
  • processin can mean performing the steps of an INC assay to detect the presence of a target protein by contacting the protein with an antibody that specifically binds to the protein, under appropriate conditions wherein the antibody specifically binds to the target protein and detecting the antibody bound to the target ⁇ e.g., by detecting a calonmetric signal ⁇ wherein detection of the signal is indicative of the presence of the target protein, and lack of signal detection is indicative of the absence of the target protein.
  • Such assay steps are well-known to those of skill in the art. Typical IHC and ISH procedures are presented in the Example section.
  • a pre-determined dataset for the eel! array and related panel of assay targets must be established.
  • the pre-determined dataset will be stored and presented on paper media or on digital media.
  • assays to detect every member included in the pane! of assay targets wiil be performed separately with relevant test reagents on the quality control DCi array devices.
  • To establish a dataset for the 26-member core panel of assay targets by IHC individual assays using antibodies to each member included in the panel wiil be done on separate cell array devices accordi g to the protoco! outlined in Example 2.
  • Ail cells in a quality control cell array wiil react with the test reagents.
  • a fraction or a!i cells in some ceil dots will produce positive signals, while aii celis in some other ceil dots wiil produce negative signal
  • positive signal refers to a signal produced by the test reagents against a specific assay target of interest that stands above the background signal and is readily distinguishable from the nonspecific background signal by human eyes and machines.
  • negative signal refers to a signal produced by the test reagents against a specific assay target of interest that is not distinguishable from the nonspecific background signal by human eyes and machines.
  • the signals will be recorded and analyzed, and given a digital or numerical indicator. For example, highly positive signals may be converted to high pixels , , or be given a high number of 'V signs.
  • subcellular localization can also be revealed and recorded for every member included in the pane! of assay targets in addition to signal strength which may reflect expression level of the target in celis.
  • a pre-determined dataset is established.
  • the dataset may be stored on paper media, in forms of, but not limited to, a table, a chart, or a collection of pictures.
  • the dataset can aiso be stored and presented by a digital medium, such as a computer monitor and a CD/DVD.
  • a digital medium such as a computer monitor and a CD/DVD.
  • Another embodiment encompasses an assay method for validation of the subcellular localization of a target molecule in a biological sample.
  • the method comprises simultaneously processing the bioiogical sample and a quality control cell array in the IHC or ISH assay as described above, The processing results in a detectable signal generated by the presence or absence of the target molecule in specified subcellular location in ceils in the biological sample and in the cells in quality control ceil array.
  • the subcellular location of detectable signal generated from the target molecule in the biological sample is compared with the location of the signal generated from the target molecule in a ceil spot in the quality control cell array,
  • Subcellular localizations inciude but not limited to, nucleus, cytoplasm, cytoplasm membrane, and nuclear membrane.
  • Expression level of a specific target and its subcellular localization is fixed for a specific cell line included in the quality control cell array and can be unambiguously determined and documented during designing end making of the quality control cell array. Result of an assay procedure on a biological sample as well as on the quality control cell array against a specific target molecuie can then be compared with the pre-determined and pre- documented subcellular localization and level of expression of this specific target molecule in cells included in the quality control ceil array.
  • the assay procedure is successful, and the result from the biological sample in the test can be reported, if the assay result generated on the quality control ce!i array device does not match the pre-determined data for the specific target, the assay procedure is in question, and the result from the biological sample in the test cannot be reported.
  • a commercial kit comprising a cell array quality control device, information for a panel of assay targets that can be definitively controlled, a pre-determined and pre- documented data set for expression level and subcellular localization of every member in the panel of assay targets in ever spots of cells included in the quality control ceil array.
  • Expression level can be presented quantitatively by digital pixels or semi-quantitatively by number of signs. For example, “+++” indicates highly positive, “++” indicates positive, “+” indicates low positive, “+/*” indicates borderline, and indicates negative,
  • a table of expression level of each assay target in each ceil spot or a computer fiie or a printout of image results of expression level of each assay target in each ceil spot acquired by scanning or digital imaging will be included,
  • Example 1 Selection of cell lines to be included in the quality control cell array is closely related the pane! of assay targets to be controlled.
  • 11 cell lines are selected to make a 11-dot quality control eel! array, including cell lines He!a (cervix adenocarcinoma), LS174T (colorectal adenocarcinoma), SW480 (adenocarcinoma, coiorectal), 8T474 (breast ductal carcinoma), MCF- 7 (breast carcinoma), 22Rvl (prostate carcinoma), Ragi (8 cell Burkit s lymphoma), HuT 78 ⁇ T cell lymphoma), Jurkat (T cell leukemia), SK-UT (sarcoma), SW1353 (sarcoma), and CaCl
  • Immunohistochemistry In this Example a biological sample (including quality control cell array) is treated with antibodies (primary and secondary), treated for chromogen color development, and finally counter-stained.
  • deparaffinization When paraffin-embedded tissue sections are used, deparaffinization must be performed, typically by a 2-minute immersion in xyiene for 5 rounds, 100% alcoho! twice, 95% alcohol once to rehydrate and then air-drying at room temperate for 5 minutes,
  • samples are washed with PBS.
  • Predicted primary antisera or antibodies (approximately 150-200 .mu.L) are applied to each sample.
  • the samples are incubated with the antisera or antibodies for 2-4 hours at room temperature or incubated in a humidity chamber at 4Q.degree, C, for 2 hours or may be incubated in a humidity chamber at room temperature overnight.
  • samples on slides are washed in a staining dish with PBS three times.
  • Prediluted secondary antibody ⁇ approximateiy 150-200 .mu.L ⁇ is applied into each sampie on slide. This is incubated for 30 minutes at 40o C in a humidity chamber. After incubation the samples are washed in a staining dish with three changes of PBS, D. Removal of endogenous peroxidase activities
  • the ABC complex ⁇ Vector laboratories inc., Buriingame, Calif, ⁇ is diluted to its working concentration using PBS.
  • the working concentration (approximately 150-200 .mu,L) is applied to each sample on slide.
  • Incubation with ABC solution is in the humidity chamber at 40" C for 30 minutes. After incubation samples on slides are washed in a staining dish with 3 changes of PBS,
  • DAB solution is prepared by adding 100 mg DAB to 100 ml PBS and adding 50 .mu.L of 30%
  • Samples on slides are immersed in Harris's hematoxylin for 10-50 seconds and washed by dipping into deionized water for three changes, and then immersed in 0.2% ammonium hydroxide solution for 30 seconds and washed by dipping in deionized water for 3 changes. They are dipped into 95% ethano! for two changes of 2 minutes each, followed by dipping into 100% ethanoi for 2 changes of 2 minutes each, and finally the slides are cleared by dipping into two changes of xylene for 2 minutes each.
  • biological samples as well as the quality control cell array (hereafter also refers to as sample) on slides are hybridized with biotin or digoxigenin labeled probes and reacted with anti-biotin or anti-digoxigenin antibody. The samples are then stained.
  • Samples on siides are deparaffinized by placing with four changes of xylene for 5 minutes each, two changes of 100% ethanoi for 1 minute each and two changes of 95% ethanoi for 1 minute each, The deparaffinized tissue section slides are then cleared and washed with detonize water with RNase Block (BioGenex, San Ramon, Calif,).
  • Hybridization solution containing a biotiny!ated or digoxigenin labeled oligonucleotide probe is placed on each sample on slide. This requires approximately 50-100 .mu A of solution. Siides are placed in an oven or on a heating biock at 95° C. for 8-10 minutes to denature the nucleic acids. This step eliminates hair-pin loops or folding back of mRNA sequences. After the denaturation step, the slides are incubated in a humidity chamber at 45° C. overnight. Following the hybridization step, the slides are washed in a staining dish with 2.times,SSC (standard saline citrate) at 37" C, for 5 minutes followed by a wash with 1 x SSC at 37° C for 5 minutes. This is followed by a 30 minute wash in 0,2 x SSC at 60° C. Finally the slides are washed with 2 changes of PBS for 2-5 mi utes each.
  • SSC standard saline citrate
  • Slides are placed vertically into a staining dish with 500 ml of 5% mixed normal goat and horse serum at room temperature for 20 minutes.
  • Prediluted mouse anti-biotin or mouse anti- digoxigenin antibody (150-200 ,mu.L) is applied to each sample on slide.
  • the samples on slides with antibody are incubated in a humidity chamber at 40° C for 2 hours. After incubation with the anti-biotin or anti-digoxigenin antibody, the samples on slides are washed in a staining dish with three changes of PBS,
  • Prediluted secondary antibody (approximately 150-200 ,mu.L ⁇ is applied into each sample on slide and incubated for 30 minutes at 40" C in a humidity chamber. After incubation the samples on slides are washed in a staining dish with three changes of PBS,
  • the ABC complex is diluted to its working concentration using PBS.
  • the working concentration (approximately 150-200.mu.L) is applied to each sample on slide and incubated in the humidity chamber at 40 fJ C fo 30 minutes. After incubation the samples on slides are washed in a staining dish with 3 changes of PBS.
  • DAB solution is prepared by adding 100 mg DAB to 100 ml PBS and adding 50 .mu.L of 30% H;0:;. Approximately 150-200 .mu.L of the DAS solution is added to each sample on slide. Color devebpment can be monitored by viewing the sample on slide with DAB under a microscope. A colored precipitate will form at the site of positive cells. Color begins to appear after 2-5 minutes, usually reaching sufficient development within 10 minutes, but a 20-30 minute incubation may be necessary for weakly stained samples. To sto development, samples on slides are washed in a staining dish with three changes of deionized water. I. Counterstaining
  • Samples on slides are immersed in Harris's hematoxylin for 10-50 seconds and washed by dipping into deionized water for three changes.
  • the slides are then immersed in 0.2% ammonium hydroxide solution for 30 seconds and washed by dipping in deionized water for 3 changes.
  • the slides are then dipped into 95% ethanoi for two changes of 2 minutes each, followed by dipping into 100% ethanoi for 2 changes of 2 minutes each, and finally the slides are cleared by dipping into two changes of xylene for 2 minutes each.

Abstract

Dispositif de contrôle qualité d'une matrice de cellules pour analyse pathologique, son procédé de fabrication et son utilisation. Des cellules cultivées d'une pluralité de types de cancers et d'origines sont placées sur des substrats solides au format matrice basse densité représentant un large éventail d'indicateurs positifs et négatifs pour procédés de dosages immunologiques et moléculaires tels que l'immunohistochimie, l'immunocytochimie, et l'hybridation in situ.
PCT/US2010/059205 2010-12-07 2010-12-07 Dispositif de contrôle qualité d'une matrice de cellules pour analyse pathologique WO2012078138A1 (fr)

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CN201080034906.5A CN103347574B (zh) 2010-12-07 2010-12-07 病理学形态分析的细胞阵列质量控制装置

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CN104931699A (zh) * 2015-05-29 2015-09-23 北京海思特临床检验所有限公司 骨髓涂片异常淋巴细胞染色试剂盒及其使用方法
WO2016049286A1 (fr) * 2014-09-24 2016-03-31 Geisinger Health System Programme de gestion de la qualité appliqué à l'immunohistochimie utilisant des lignées cellulaires en culture pour produire des blocs de puces tissulaires (tma)
EP3258264A4 (fr) * 2015-02-10 2018-01-10 Konica Minolta, Inc. Procédé de quantification de matériel biologique, système de support pour diagnostic pathologique et programme
CN110753846A (zh) * 2017-06-15 2020-02-04 日光石科技有限公司 免疫组织化学抗原成像标尺外推方法
WO2020075137A1 (fr) 2018-10-12 2020-04-16 Sunstone Scientific Ltd. Lame d'enregistrement du processus de coloration et procédé d'utilisation associé

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WO2016049286A1 (fr) * 2014-09-24 2016-03-31 Geisinger Health System Programme de gestion de la qualité appliqué à l'immunohistochimie utilisant des lignées cellulaires en culture pour produire des blocs de puces tissulaires (tma)
EP3258264A4 (fr) * 2015-02-10 2018-01-10 Konica Minolta, Inc. Procédé de quantification de matériel biologique, système de support pour diagnostic pathologique et programme
CN104849465A (zh) * 2015-05-29 2015-08-19 广州华弘生物科技有限公司 肿瘤血清标志物ca153、cea、hcg乳腺癌三联诊断试剂盒及其制备方法
CN104931699A (zh) * 2015-05-29 2015-09-23 北京海思特临床检验所有限公司 骨髓涂片异常淋巴细胞染色试剂盒及其使用方法
CN104849465B (zh) * 2015-05-29 2016-06-29 广州华弘生物科技有限公司 肿瘤血清标志物ca153、cea、hcg乳腺癌三联诊断试剂盒
CN110753846A (zh) * 2017-06-15 2020-02-04 日光石科技有限公司 免疫组织化学抗原成像标尺外推方法
CN110753846B (zh) * 2017-06-15 2024-03-26 深圳市诺高实验器材有限公司 免疫组织化学抗原成像标尺外推方法
WO2020075137A1 (fr) 2018-10-12 2020-04-16 Sunstone Scientific Ltd. Lame d'enregistrement du processus de coloration et procédé d'utilisation associé
JP2022520536A (ja) * 2018-10-12 2022-03-31 シンセン ピーアールエス リミテッド 染色用のプロセス記録スライド及びそれを使用する方法
EP3847496A4 (fr) * 2018-10-12 2022-06-22 Shenzhen PRS Limited Lame d'enregistrement du processus de coloration et procédé d'utilisation associé

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