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WO1998020353A1 - Cytoperfusion de specimens tissulaires - Google Patents

Cytoperfusion de specimens tissulaires Download PDF

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Publication number
WO1998020353A1
WO1998020353A1 PCT/US1997/019871 US9719871W WO9820353A1 WO 1998020353 A1 WO1998020353 A1 WO 1998020353A1 US 9719871 W US9719871 W US 9719871W WO 9820353 A1 WO9820353 A1 WO 9820353A1
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WO
WIPO (PCT)
Prior art keywords
specimen
tissue
tissue specimen
film
support
Prior art date
Application number
PCT/US1997/019871
Other languages
English (en)
Inventor
Charles M. Mcgrath
Jennipher L. Grudzien
Original Assignee
Grace Bio-Labs, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Grace Bio-Labs, Inc. filed Critical Grace Bio-Labs, Inc.
Priority to AU54273/98A priority Critical patent/AU5427398A/en
Publication of WO1998020353A1 publication Critical patent/WO1998020353A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N1/312—Apparatus therefor for samples mounted on planar substrates

Definitions

  • This invention concerns histological preparation and examination of tissue specimens for diagnostic purposes.
  • the invention is directed to cytologic and immunofluorescent examination of pathological specimens for diagnostic purposes.
  • Microscopic examination of tissue specimens for histopathologic purposes has gready enhanced the effectiveness of medical treatments for a variety of illnesses, because microscopic examination permits a more specific diagnosis of a pathological condition.
  • a diagnosis is made on the basis of cell morphology and staining characteristics.
  • Tumor specimens for example, can be examined to characterize the tumor type and predict whether the patient will respond to a particular form of chemotherapy.
  • the microscopic examination and classification of tumors has improved medical treatment, the microscopic appearance of a tissue specimen stained by standard methods (such as hematoxylin and eosin) can only reveal a limited amount of diagnostic information.
  • Recent advances in molecular medicine have provided an even greater opportunity to understand the cellular mechanisms of disease, and select appropriate treatments with the greatest likelihood of success.
  • Some hormone dependent breast tumor cells for example, have an increased number of estrogen receptors on their cell surfaces that indicate that the patient from whom the tumor was taken will respond to certain anti-estrogenic drug treatments.
  • Other diagnostic and prognostic cellular changes include the presence of tumor specific cell surface antigens (as in melanoma), the production of embryonic proteins (such as ⁇ -fetoprotein in liver cancer and carcinoembryonic glycoprotein antigen produced by gastrointestinal tumors), and chromosomal abnormalities (such as the presence of oncogenes).
  • a variety of techniques have evolved to detect the presence of these cellular abnormalities, including immunophenotyping, in situ hybridization, flow cytometry, and DNA amplification using the polymerase chain reaction (PCR).
  • One approach to molecular diagnosis has been to homogenize cells from a tissue specimen, to expose the cellular contents to a liquid environment in which analysis of the biochemical markers can be performed.
  • This approach has been used to detect changes in the concentration of epidermal growth factor (EGF) or estrogen receptors (ER) in breast cancer.
  • EGF epidermal growth factor
  • ER estrogen receptors
  • a drawback of this method has been that it destroys the cytocoherence of the cells, which can remove important information about the tumor.
  • Another problem with this quantitative assay is that malignant cells often proliferate dramatically, while the cell surface marker proliferation is more subtle. The greater proliferation of the cellular compartment can overwhelm the more subtie increase in the cell surface markers, thereby obscuring quantitative information about marker concentration.
  • a probe is any molecule that specifically binds to a nucleic acid sequence or protein of interest, and which can be labeled so that the required targets can be detected.
  • Probes for nucleic acid sequences are radiolabeled or chemically tagged oligonucleotides of complementary sequence to the DNA or RNA sequence that is diagnostic of a genetic change associated with a tumor (such as the Philadelphia chromosome in chronic granulocytic leukemia).
  • a monoclonal antibody can be designed to recognize antigens that are biochemical markers (such as the Her-2 and epidermal growth factor (EGF) receptors in breast cancer).
  • biochemical markers such as the Her-2 and epidermal growth factor (EGF) receptors in breast cancer.
  • the antibodies may undergo a chromogenic immunochemical reaction that is proportional to the concentration of the biochemical marker, such that the concentration of the marker on the cell can be colorometrically quantitated.
  • the reaction may either be direct (fluorochrome directly conjugated to the primary antibody) or indirect (two- stage procedure in which a secondary fluorochrome conjugated antibody is directed against the primary antibody).
  • Such basic techniques of immunohistochemistry include immunofluorescent staining methods and enzyme conjugation procedures) are disclosed in DeLellis, Diagnostic Immunohistochemistry, Masson Publishing, 1981, chapter 1 and 2.
  • cytocoherent transfer of a tissue specimen can be achieved by thawing a frozen sample on a membrane impregnated with a surfactant. Cytocoherent transfer is a transfer that preserves the location of the target cell solute (such as the location of a protein receptor in the cell membrane) with accurate planar definition relative to cellular microanatomy and organdies with which the solute associates.
  • Lyophilization of pathologic specimens for subsequent analysis has also been discussed in the scientific literature.
  • An example is Stumpf and Roth, "High Resolution Autoradiography with Dry Mounted, Freeze-Dried Frozen Sections", /. of Histochemistry and Cytochemistry; 14:274-287 (1966), in which frozen sections of tissue are freeze dried on emulsion coated glass slides for autoradiography.
  • Another example is U.S. Patent No. 5,059,58, in which mammalian cells are lyophilized after being exposed to trehalose to produce a cell pellet that, when rehydrated, retains optimal physiological characteristics. Cells prepared in this fashion are disaggregated, and not cytocoherent.
  • U.S. Patent No. 4,681,853 describes a method of perfusing reagents into a chamber holding a membrane to which nucleic acids are bound, for the purpose of treating the surfaces of the membrane.
  • U.S. Patent No. 5,312,731 shows perfusion of cytotoxic drugs through a cell aggregate suspended in a perfusion chamber.
  • Immunocytochemical reagents are then introduced into the chamber where a reaction between the reagent and tissue specimen can occur.
  • significant obstacles still remain to the routine use of probes in the examination of cytological specimens.
  • a typical colorirnetric immunoassay may require 4-5 hours, including 15-30 minutes for retrieval of the specimen, 30-60 minutes for fixation, 60-90 minutes for exposure to the primary antibody, 15 minutes to wash out the primary antibody, 30-60 minutes to expose the tissue to a secondary antibody, linker or enzyme that recognizes the primary antibody to produce a colorirnetric change, followed by another 15 minute wash.
  • In situ hybridization of an oligonucleotide probe to a tissue specimen typically includes even more steps and longer incubations. These prolonged treatments reduce the efficiency of the assay and increase its cost.
  • Another object of this invention is to provide such an assay that provides reliable and more accurate results that are capable of quantitative interpretation.
  • the present invention includes a method of preparing tissue specimens for microscopic examination, by mounting a tissue specimen on a porous support that retains cytocoherence of the specimen.
  • the tissue specimen (which may for example be a thin section, touch prep, body fluid smear, fine needle biopsy specimen, cell culture, or a biological fluid such as blood or urine) is then lyophilized in a cytocoherent form on the porous support under conditions that form microchannels in the specimen, so that a reagent can be perfused through the specimen and support.
  • Perfusion of the reagent such as a probe
  • the porous specimen permits a rapid and effective reaction to occur between the specimen and probe.
  • Subsequent steps such as washing the specimen or introducing secondary antibodies, can also be performed by perfusion.
  • Perfusion of reagents and washing solutions substantially reduces the reaction time and cost of the assay, improves the quality of the microscopic image, and enhances signal accuracy of colorirnetric and other indicators used in quantitative studies.
  • the specimen is lyophilized below a eutectic temperature of the specimen.
  • the eutectic temperature is the initial melting point of the specimen, at which water in the frozen specimen begins to liquefy (and electrical conductivity increases). Lyophilization below this temperature allows liquid to be sublimed from the solid to the gaseous phase, to form pores in the specimen through which probes and other reagents can be perfused. Lyophilization is performed under conditions that create a vertical temperature gradient, and form vertical microchannels in the specimen, to enhance the flow of reactants through the specimen. The formation of such microchannels has previously been considered an undesirable artifact, but the present invention has disregarded that teaching to arrive at the improved perfusion process.
  • the reagent is a diagnostic antibody, which is perfused through the specimen and support at a pressure of about lOpsi, and a flow rate of about 2 ml/cm 2 /min. Negative pressure (vacuum) as well as positive pressure can be used to perfuse the reagent through the porous specimen.
  • the specimen may be lyophilized under conditions that form pores of about 1 ⁇ diameter in the specimen, and a pore density of at least about 0.6 x 10 8 pores/cm 2 of tissue.
  • the porous support can be a composite film that includes a nitrocellulose surface layer to which the tissue specimen is adhered, and a microporous subjacent layer that has a light transmittance which is at least 60% the hght transmittance of glass.
  • the porous subjacent layer may be a microporous nylon, ceramic or cellulose material.
  • the support can be impregnated with a detergent (such a sodium dodecyl sulfate or sodium lauryl sulfate) that improves adherence of the specimen to the support.
  • a detergent such a sodium dodecyl sulfate or sodium lauryl sulfate
  • An excipient may be added to the specimen prior to lyophilization, to normalize the eutectic temperature of the specimen at a desired process temperature, so that reproducible, uniform lyophilization will occur in specimens of varying compositions.
  • the reagent may be perfused through the specimen and composite film by attaching a sealed chamber to a surface of the support, and introducing the reagent in liquid form into the sealed chamber and through the specimen and film under pressure (either positive pressure or vacuum pressure).
  • the composite film limits a rate of perfusion of the reagent, i.e. the flow rate through the composite film is lower than the flow rate through the tissue specimen. This flow limitation helps prevent uneven flow through the specimen caused by areas of reduced specimen density (such as artifactual separations), and avoids damaging the tissue with excessive flow rates.
  • the specimen (having a thickness of less than about 20 ⁇ ) may be lyophilized at a temperature of -70 to -30°C, for example -70 to -40°C, more specifically -40 C C, to produce a pore size of about 1 ⁇ or less. This pore size is preferred because the pores are not readily visible when microscopic images are viewed at a magnification of 400X or less (e.g. 250-400X).
  • the specimen is also lyophilized by freezing the specimen rapidly, for example at a rate of greater than 5°C, or 20°C, 50°C or 100°C per second to promote the formation of vertical microchannels.
  • the method includes adhering a thin tissue specimen to a dry nitrocellulose film mounted to a porous support having an optical transmittance that is at least 60% the transmittance of glass.
  • the porous support may be microporous nylon (such as NytranTM) having pores with a diameter of about 0.1-0.45 ⁇ .
  • the tissue specimen is then lyophilized under conditions that maintain its cytocoherence and form vertical microchannels in the specimen, while retaining cytocoherence of the specimen.
  • a liquid reagent (such as a monoclonal antibody for immunophenotyping) is then perfused under pressure through the tissue specimen and porous support to react the liquid reagent with the tissue specimen.
  • the specimen is then washed to remove unreacted reactant, by perfusing a washing liquid under pressure through the tissue specimen and porous support.
  • a secondary antibody may then be introduced (for example to perform an indirect assay), where the secondary antibody recognizes the primary antibody that has adhered to proteins of interest to provide a colorirnetric, fluorescent or radioactive signal. This step may be followed by a second washing step to remove excess secondary antibody from the specimen.
  • the tissue specimen is lyophilized under conditions that form a lyophilized tissue specimen having pores of a diameter of less than about 1 ⁇ and a tissue porosity of less than about 50% , and the porous composite film support is less porous than the lyophilized tissue specimen so that a rate of flow of reagent through the tissue specimen is limited by a rate of flow of the reagent through the porous support. Lyophilization of the specimen also occurs in the presence of an effective amount of a normalizing agent to normalize a eutectic temperature of the specimen, where the specimen is frozen at a rate of at least 20 °C per second, and sublimation occurs for at least 1 minute at a temperature of -40 to -70°C.
  • the liquid is perfused through the tissue specimen and porous composite film under a pressure of less than about 10 psi, and at a flow rate of less than about 2 ml/cm 2 /sec, by introducing the liquid into the chamber under pressure.
  • the method of preparing the tissue specimen for microscopic examination includes providing a thin tissue specimen having a thickness of less than about 20 ⁇ , wherein the tissue specimen is impregnated with a sufficient concentration of an excipient to normalize a eutectic temperature of the tissue specimen to a desired temperature of about -40° C.
  • Suitable excipients include an alcohol (such as t-butyl alcohol, or high molecular weight alcohols such as polyvinyl alcohol).
  • a composite film support which includes a nitrocellulose surface film having a thickness of about 5 ⁇ , a pore size of 0.01-0.8 ⁇ and a porosity of less than about 50% , adhered to a porous nylon base, wherein the nylon base has a pore size of about 0.2 ⁇ , a thickness of about 100 ⁇ , and a hght transmittance that is at least 60% of a light transmittance of glass.
  • the nitrocellulose film is impregnated with a detergent that improves adherence of the tissue specimen to the nitrocellulose film, and the tissue specimen is placed on the nitrocellulose film in a dry condition so that the tissue specimen adheres with cytocoherence to the nitrocellulose film.
  • the tissue specimen is lyophilized by placing the composite film on a freezing platen, which creates a vertical temperature gradient in the specimen, and freezing the specimen at a rate of at least 20°C. Solid ice is sublimed from the specimen, below the eutectic point, for at least one minute at a temperature of about -40 C C, and at a pressure of about 1 x 10 ⁇ 3 mm Hg, to produce vertical microchannels in the tissue specimen.
  • a pressure chamber is then placed against the tissue specimen, to perfuse a liquid reagent containing a diagnostic antibody through the tissue specimen and composite support.
  • the liquid reagent may be perfused at a positive or negative pressure of about 10 psi, and a flow rate of about 2 ml/cm 2 /min for no more than about 1 minute, wherein the flow rate is lower through the nitrocellulose than through the tissue specimen film.
  • a washing liquid, and/or secondary antibodies can subsequently be perfused through the tissue specimen and composite support.
  • a magnified image of the tissue specimen at a magnification of about 250-400 is then examined through a microscope.
  • the amount of the antibody or other reagent that binds to the tissue can be quantitated, for example by measuring the color intensity of the specimen.
  • the invention also includes a composite support for microscopic examination of a tissue specimen, wherein the composite support includes a surface film that adheres thin tissue specimens in a cytocoherent manner, and a base to which the surface film is mounted.
  • the surface film and base are sufficiently porous to allow a reactant liquid to be perfused through the surface film and base.
  • the composite support further includes a thin tissue specimen lyophilized on the surface of the film.
  • the surface film may be a nitrocellulose film that is about 5 ⁇ thick, has a pore size of about 0.01-0.8 u and a porosity of less than about 50% , and the base has a pore size of about 0.2 ⁇ , a thickness of about 100 ⁇ , and a light transmittance that is at least 60% of a light transmittance of glass.
  • the surface film or composite support may be impregnated with a detergent, such as sodium dodecyl sulfate or sodium lauryl sulfate, that improves adherence of the tissue specimen to the film.
  • the base comprises a porous nylon material, such as NytranTM.
  • the invention also includes a system for microscopic examination of a thin tissue specimen, comprising a porous support to which the thin tissue specimen adheres in a cytocoherent fashion, and on which the thin tissue specimen can be lyophilized under conditions that form perfusion pores in the thin tissue specimen while retaining its cytocoherence.
  • the system also includes a perfusion attachment capable of being secured to the porous support, and capable of perfusing a liquid through the thin tissue specimen and porous support under pressure without disrupting the cytocoherence of the tissue.
  • the perfusion attachment may be a chamber with an annular seal for placement against the support.
  • FIG. 1 A is a top perspective view of a slide made in accordance with the present invention, with a porous composite film of the present invention incorporated into the slide, and with a tissue specimen on the composite film.
  • FIG. IB is a cross-sectional view of a composite film on a glass slide from which the composite film may be removed for perfusion.
  • FIG. IC is a cross-sectional view of a composite film on a polycarbonate slide through which a perfusion hole has been cut, so that perfusion Uquid can be introduced from a perfusion tube through the composite film without removing the film from the slide.
  • FIG. 2 is a top view of the isolated composite film and tissue specimen, after the composite film is removed from the slide.
  • FIG. 3 is a cross-section scanning electron micrograph of the composite film of FIG. 2.
  • FIG. 4 is a photomicrograph of the microporous nylon support of the film shown in FIG. 1.
  • FIG. 5 is a photomicrograph of the nitrocellulose surface of the composite support, showing the pores that occupy about 50% of the surface of the nitrocellulose.
  • FIG. 6 is a perspective view of a perfusion apparatus for introducing immunohistochemical reagents through the tissue specimen.
  • FIG. 7 is an enlarged view of the perfusion chamber of the apparatus shown in FIG. 6.
  • FIG. 8 is an exploded perspective view of a perfusion manifold for performing cytoperfusion.
  • FIG. 9 is a top perspective view of a lyophilization apparatus for lyophilizing tissue specimens in accordance with the present invention.
  • FIG. 10 is a graph showing increased washing efficiency with the perfusion technique of the present invention, as compared to diffusional washing.
  • the cytoperfusion technique of the present invention allows the cytocoherent transfer of proteins and nucleic acids (as well as other solutes) from porous pathological specimens (such as thin section surgical specimens from plant or animal origins) to composite supports, by lyophilizing the tissue specimen on a nitrocellulose layer of the support under conditions that promote formation of pores and vertical microchannels.
  • the porosity of the specimen permits a probe solution to be perfused through the specimen under pressure, while retaining microanatomic relationships within the specimen.
  • the advantage of cytoperfusion is that it greatly reduces the time needed to obtain highly sensitive results with immunoassays. Microchanneling of the specimen increases access of reactants by exposing reaction sites and creating greater surface area within the specimen for reactions to occur.
  • a specimen that is approximately 10 microns thick is freeze dried in situ on a nitrocellulose/nylon composite film, in which the nitrocellulose component has pores that are 0.1 to 0.3 microns in diameter, and the pores of the nylon component are even larger.
  • Lyophilization conditions temperature -40 to -70°C, below the eutectic temperature, and rate of coohng at least about 1 °C per second
  • Uniformity of pore size can be promoted by applying an excipient solution (such a t-butyl alcohol) that promotes formation of eutectics by normalizing the distribution of temperatures at which ice crystal formation occurs.
  • a temperature gradient is preferably created from the bottom to the top of the specimen, so that the specimen freezes from the bottom to the top.
  • This directional freezing forms microchannels that allow perfusion of the probe reagents throughout the specimen in three dimensions.
  • the size of the channels (directional pores) is sufficiently small not to compromise resolution of cellular microanatomy when the specimen is viewed under a light microscope at 250- 400X.
  • Perfusion of the probe reagents is performed at a pressure of 5-10 psi with a flow rate of about 2-5 ml/min/cm 2 through the tissue specimen.
  • FIGS One embodiment of the composite support 20 of the present invention is shown in FIGS
  • a circle of microporous nylon membrane 19 (NYTRAN from Schleicher & Schuell) having a 22 mm diameter is placed on slide 21.
  • Polyester tape mask 22 is prepared by cutting an 18 mm diameter cut-out using a punch.
  • the mask 22 has an adhesive undersurface that is applied to the surface of slide 21 , with the cut-out in the mask 22 centered on the nylon membrane 19.
  • a layer 26 of nitrocellulose polymer is then spun cast in a conventional fashion on to the surface of nylon membrane 19 and mask 22, so that the nitroceUulose overlaps the borders of mask 22 and occupies a circular area having a diameter that is about the same as the diameter of the nylon membrane 19.
  • the Uquid nitroceUulose polymer from which the layer 26 is formed may, for example, be made by purchasing raw nitroceUulose materials from Hercules- Aqulon (Wilmington DE) 68.82 g RS 3/4sec, plus 68.82 g RS 18-25cps, dissolved into a mixture of 196.32 g n-butanol (purchased from Fisher Scientific, cat# A383-4) and 7.2 g isopropanol (Fisher Scientific cat#A16P-4), and 23.10 g lauryl sulfate solution (prepared by dissolving 6.46 g of lauryl sulfate (Sigma Ultragrade cat# L-6026) in de-ionized water and stirred gently to dissolve the lauryl sulfate without foaming).
  • the nitrocellulose polymer solution can be stored at room temperature untU spin casting at 1000 rpm for 70 seconds. After spin casting, the composite film is cured at room temperature for 10 minutes, fo
  • a perforation 24 is placed through layers 26, 22 and 19, and the perforation extends in the mask layer away from the composite film to form a pull-away tab 28 that can be puUed to remove the composite film from slide support 21.
  • the composite film that is removed includes mask 22 and underlying nylon layer 19 to which mask 22 is adhered, as weU as nitroceUulose layer 30 which is supported by mask 22.
  • a tissue specimen 32 on the nitroceUulose layer 30 the composite membrane and tissue specimen are placed in a perfusion device, such as that shown in FIGS. 6 and 7.
  • FIG. IC An alternative embodiment of the composite membrane is shown in FIG. IC, which is similar to the embodiment shown in FIG. IB, so that Uke parts have been given Uke reference numerals.
  • the slide is made of polycarbonate, and a cylindrical hole 23, about 13 mm in diameter, is provided through the slide 21.
  • the hole 23 is fiUed with a porous fiUer (such as a porous glass plug), which provides a porous opening through which perfusion Uquid can be introduced, wlule still providing a thermal conductance similar to the surrounding sUde material 21.
  • the nitroceUulose and Nytran layers are not separated from the slide 21 by puUing tab 28 away from sUde 21 to provide an isolated composite film 31 consisting of the Nytran layer 22 and nitroceUulose layer 30.
  • the tissue specimen 32 can be perfused in situ on the sUde 21 because of the hole 23 through the slide.
  • a perfusion tube 33 is shown in FIG. IC, which can seat on nitrocellulose layer 30 around specimen 32 to perfuse the specimen.
  • FIG. 6 A simplified perfusion chamber apparatus 50 for use with the composite film of FIG. IB is shown in FIG. 6, and an enlarged view of the perfusion chamber 52 of the apparatus is illustrated in FIG. 7.
  • the chamber 52 is a Sweeny filter assembly having a funnel shaped base 54 with an externally threaded neck 56 that mates with an internally threaded cap 58.
  • the composite film 31 with its adhered specimen 32, is placed on a support screen 59 that seats on the neck 56, and the gasket (O-ring) 34 seats on the composite film 31 (around specimen 32) to further seal the chamber 52 when cap 58 is mated with the threaded neck 56.
  • Chamber 52 is connected to a sihcone rubber coupUng 60, which is in turn clamped (with clamp 62) to the bottom of a 5 ml graduated pipette 64.
  • the top of pipette 64 is in turn attached to a sihcone rubber coupUng 66, which is clamped with clamp 68 to a gas connector 70.
  • Nitrogen gas tubing 72 is connected to a source of pressurized gas (not shown), and the flow of gas can be regulated by a rotatable on/off valve 74.
  • the apparatus 50 is mounted on a ring support stand 76 with ring stand clamps 78, 80 in the conventional fashion.
  • a reagent liquid such as a liquid containing a monoclonal antibody
  • a reagent liquid is placed in pipette 64, and perfused through composite film 30 by introducing nitrogen gas (at about 10 psi) into tubing 72.
  • nitrogen gas at about 10 psi
  • the gas pressure forces reagent Uquid from the pipette 64, through composite film 30, and out of the filter assembly 52.
  • FIG. 8 is a modified filter manifold that can be obtained from MUUpore Corporation of Bedford, MA, USA.
  • the cytoperfusion apparatus includes a cylindrical reservoir chamber base 102 having a central positioning/locking post 104 that extends above base 102 and terminates with an externally threaded end 106.
  • a reagent funnel 108 tapers to an outlet 110, and has an annular flange 112 around its top, peripheral to a series of outlet orifices 114.
  • a tissue disc platform 116 which seats on funnel 108 supports a removable rigid plastic disc 117 with peripheral cut-outs in which are mounted the composite films 31.
  • a housing 118 with a series of cyUndrical reagent cups 120 seats on tissue disc platform 116 with the cups 120 ahgned with the composite films 31, and a disposable rotary reagent tray 122 with a series of complementary cyUndrical inserts 124 that fit within cups 120.
  • a reagent (such as lyophUized monoclonal antibody) is adhered to the inner cylindrical surface of inserts 124.
  • a coyer 126 fits on chamber base 102 above tray 122, and is secured in place by lock nut 128.
  • Tubing 130 communicates with the interior of apparatus 100 through cover 126.
  • support disc 118 is placed on platform 116 with lyophilized tissue specimens on one or more of the composite films 31.
  • the cytoperfusion manifold is assembled by placing the funnel 108 in chamber base 102, with platform 116 seated on funnel 108. Housing 118 is seated on platform 116, with reagent cups 120 aligned over composite films 30, and the cyhndrical inserts 124 of the tray 122 are inserted into the complementary cups 120. Cover 126 is secured in position with lock nut 128, and pressurized Uquid is introduced into apparatus 100 through tubing 130. The liquid solubilizes the lyophUized reagent in inserts 124, and perfuses the reagents through the tissue specimens on composite films 31. The perfusion Uquid is collected by funnel 108, and coUected in chamber 102.
  • the invention also includes an apparatus 130 (FIG. 9) for freezing a tissue specimen below its eutectic temperature, and providing a vertical temperature gradient that gives directionaUty to the pores (vertical microchanneling).
  • Apparatus 130 includes a lyoplulization chamber 132, a clear plastic cover 134, a heat sink chamber 136 below chamber 132, a series of cooUng fins 133 extending inparaUel rows through heat sink chamber 136 to circulate cool air, a cooling chamber 138, below heat sink chamber 136, into which liquid nitrogen is introduced, and a solid base plate 140.
  • Six identical platens 142 are arranged in two rows within enclosure 132, and each platen has a cross-shaped recess 144 in its surface. The long arm 144a of the cross accepts an elongated slide, whUe the short arm 144b permits the slide to be picked up from the recess.
  • a Uquid nitrogen inlet 150 into cooUng chamber 138 communicates with a liquid nitrogen line 152, and the flow of liquid nitrogen into cooling chamber 138 is controlled by valves 154, 156.
  • a vacuum manifold 158 communicates with lyophilization chamber 132, and leads to vacuum valve 159 and a vacuum condenser line 160, to establish a vacuum in the lyophUization chamber 132.
  • Vacuum vent Unes 162 (controlled by vent valve 164) communicate with lyophilization chamber 132 to open chamber 132 to atmospheric pressure once lyophUization has been completed.
  • liquid nitrogen is introduced into cooUng chamber 138 through inlet 150 to precool platens 142 to -40° to -70°C.
  • a composite film sUde (like the sUde 20 in FIG. IB), with a tissue specimen 32 on the composite film, is then placed in each of the recesses 144a on the surface of platen 142.
  • Cover 134 is secured in place to seal lyophilization chamber 132, and the interior of lyophUization chamber 132 is evacuated to a pressure of approximately 100 miUitorr. After one minute of lyophilization at this temperature and pressure, the temperature of the lyophUization chamber is increased over 5 minutes to +25°C.
  • the vacuum in lyophUization chamber 132 is then vented to atmosphere through vent 164, and the chamber 132 can be opened to remove the sUde which contains the now lyophUized specimen.
  • Perfusion of immunocytochemical reagents through a lyophUized tissue specimen prepared in accordance with the present invention overcomes problems of poor probe binding rates (signal intensity) and non-specific probe turnover rates (noise intensity).
  • Analyte (such as a tissue bound receptor of interest) is immobiUzed in the tissue by adherence to the nitrocellulose surface, hence the probe can more easily interact with the analyte in the lyophilized tissue than in solution.
  • the use of the specialized microporous films of the present invention overcomes the problem of diffusional loss of analytes by providing a relatively permeable soUd phase reaction matrix to bind soluble analytes.
  • the nitrocellulose specimen support permits vertical (capUlary) transfer of soluble analytes from the specimen to the underlying film, to achieve cytocoherent transfer. Cytocoherent transfer controls diffusional loss by immobilizing analyte on a high-capacity binding matrix, which circumvents the counterproductive and antagonistic practices of "masking" analyte by over fixing the specimen and then "unmasking" the analyte by hydrolytic or other means.
  • the binding matrix stiU presents analyte in solid phase, where reactions based on coUision probabiUties proceed more rapidly than in solution, but the regularity of the matrix and its porosity creates a reaction environment in which analyte in solid phase is uniformly more accessible to perfused probe reactants.
  • the present invention particularly increases precision and sensitivity of immunoassays where lyophUization occurs under conditions that promote eutectic freezing and sublimation of crystaUine ice, and where pore size is controUed within certain ranges.
  • the maximum preferred pore size corresponds with the resolving power of microscope objectives used. When the maximum magnification used is 400X, a pore size of approximately 1 ⁇ or less is preferred.
  • the flow rate projected from the Darcy equation for composite film supports was 0.5-2ml/min cm 2 .
  • Perfusion rates of reagents through isolated discontinuous biological specimens e.g. dispersed cells or coherent tissue with cracks or holes
  • the composite films of the present invention achieve these differential flow rates, by casting nitrocellulose films that have smaller pores, or less porosity, than the lyophUized tissue specimen.
  • the present examples use specimens that are at least 6 ⁇ thick, which achieve this flow limitation.
  • the porosity of the lyophilized tissue specimen can also be altered to affect the rate of perfusion.
  • Porosity of the specimen can be varied by manipulating the (re)-freezing temperature during lyophilization. Generally, lower temperatures (-40 to -70°C) provide smaUer pores in the lyophUized specimen than higher refreezing temperatures (such as -30 °C).
  • a rectangular (20mmX20mm) sheet of nylon depth filter material ("0.2um" pore size Nytran, lot 16/9, Schleicher & SchueU, Keene, NH) was affixed to a polyester liner (M57) with a clean-release adhesive (Furon, Inc. , New Haven, CT), as described in connection with FIG. IB above.
  • a nitroceUulose cytocoherent transfer film was then polymer cast onto the masked filter.
  • the composite film was soaked in a detergent solution (SDS or SLS) which was dried in the material at 50 °C before use. FoUowing lyophilization, the composite films were peeled from supports and trimmed by means of a swivel-blade X-acto knife, leaving a 1.5 mm mask margin around the Film for handUng and support.
  • EXAMPLE 3 Immunocytochemistry Techniques An indirect assay, employing primary antibody and either biotinylated secondary (horse anti-mouse IgG) antibody/ ABC-AP, or secondary (goat anti-mouse) antibody-AP, and BCIP/NBT- based color detection, was routinely used.
  • Conjugated secondary antibodies and color development reagents were aU obtained from Vector Laboratories (Burlingame, CA). Two primary antibodies were used: One mouse monoclonal against Her-2/neu (mAb-1, Corning Diagnostics, Alameda, CA) and a polyclonal rabbit antibody to EGFR (Ab-4; Oncogene Science, Manhasset, NY).
  • the perfusion based assay was performed with a vertical volumetric column as shown in FIG. 6, which was constructed from a 1 (or 5) ml plastic pipette 64 connected to a source of dry nitrogen gas with pressure regulated in the range of 1-20 psi.
  • Composite films 30 were inserted into the column at the base, using a Sweeny Filter Holder 52 (Gelman Sciences, Ann Arbor, MI) with a 13mm diameter filter area. The total Film area exposed was 0.81 cm 2 .
  • a mask of soft (40 durometer) sUicone rubber with a 0.09 cm 2 hole was placed over the composite film to filter smaller specimen areas. Before use, fluids were pre-filtered through 0.05 ⁇ pore size Nucleopore filters (Corning-Costar, Cambridge, MA). How rate through films was determined at 20°C and monitored volumetricaUy using a stopwatch.
  • Specimens were stained with hematoxylin and eosin (H&E) for morphological examination. Isopropanol was substituted for ethanol in preparation of eosin. To prepare specimens for tight microscopy, stained specimens were dehydrated in an isopropyl alcohol series (30-100%) and composite films were cleared to transparency by filting pores with xylene (13), which has a refractive index similar to the composite film. Specimens on composite films were permanendy mounted on microscope sUdes in Accumount (Baxter SP, McGaw Park, II).
  • the resolving power of the microscope was approximately 1 ⁇ , based on the relationship between numerical apertures of the condenser and objective, and the wavelength of visible tight (22).
  • EXAMPLE 4 Perfusion Example a fresh piece of unfixed tonsti tissue, obtained as a surgical specimen, is snap frozen by submerging it in Uquid nitrogen.
  • the frozen tissue specimen is mounted on a Cryostat chuck using tissue mounting medium, and cut into a thin frozen section that is 2-10 ⁇ thick.
  • the thin section is thaw mounted on the nitroceUulose surface of a composite film (with a glass or polyester base), using the same technique used to apply a frozen section to a glass microscope slide.
  • the specimen is immediately re-frozen by placing the composite film on a platen surface of the lyophilization device (FIG. 9), in which the platens have been precooled to -40° C.
  • the lid of the lyophUization chamber is then closed, with the lid gasket pressed tightly against the opening to form a good seal.
  • a vacuum is established in the lyophilization chamber.
  • the amount of vacuum may vary depending on the specimen thickness and water content, but efficient lyophilization is usually achieved in a short cycle time at 100 mUUtorr pressure in the chamber.
  • Lyophilization proceeds at -40°C for one minute. WhUe maintaining a continuous vacuum in the chamber, the temperature of the platens is increased to +25°C over a period of 5 minutes (although the time and temperature can be varied for different specimens). At the end of this period, vacuum is maintained for 1 minute at +25°C, then the vacuum is broken. The Ud of the lyophilizer is opened, and the dried samples removed. Samples are then prepared for perfusion by separating the composite film (nitrocellulose and Nytran) from the glass or polyester support by peeting or cutting, and trimming the composite film (with adhered tissue specimen) to a diameter of 13 mm.
  • composite film nitrocellulose and Nytran
  • Each trimmed sample is placed into the Sweeny filter assembly (FIG. 6) which holds a 13 mm diameter filter, such that the specimen affixed surface is positioned away from the Sweeny filter support screen.
  • the threaded cap is used to close the assembly, and three ml of blocking solution (10% horse serum from Vector Labs, cat# S-1000) diluted in phosphate buffered saline (pH 7.5) containing 0.3% Tween-20 (TPBS) which has been pre-filtered through a 0.2 ⁇ ceUulose acetate filter is placed into a 5 ml graduated pipette.
  • the pipette is supported in a vertical orientation by a ring stand to which the pipette is clamped.
  • the top of the pipette is attached to a pressurized tank of nitrogen gas through a connector with an on/off valve and an adjustable pressure gauge set to 10 psi.
  • the lower tip of the pipette is fitted with a piece of flexible tubing with a 1/8 inch inner diameter, and the threaded portion of the Sweeny filter assembly is connected to the pipette by inserting the threaded exterior portion of the cap into the sUicone tubing.
  • Reagent flow through the specimen is started by opening the pressure valve at the top of the reagent pipette, and 2 ml of blocking solution is delivered through the specimen at 10 psi.
  • a typical specimen wUl allow reagent to perfuse through the specimen at a rate of 2 ml/min at 10 psi.
  • the surface of the specimen is constantly bathed in reagent solution and not aUowed to dry.
  • reagent flow is stopped by turning off nitrogen gas flow.
  • the pipette is temporarily disconnected from the Sweeny filter and filled with 0.5 ml of dUuted primary antibody reagent solution, such as anti-PCNA mouse antibody (Vector Labs, Burlingame, CA. , Cat#NCL-PCNA). Dilution of primary antibody is made in TPBS containing 1.0% normal horse serum (Vector Labs, Cat# S-2000). After re-connecting the Sweeny filter to the pipette, reagent flow is started again.
  • dUuted primary antibody reagent solution such as anti-PCNA mouse antibody (Vector Labs, Burlingame, CA. , Cat#NCL-PCNA).
  • Dilution of primary antibody is made in TPBS containing 1.0% normal horse serum (Vector Labs, Cat# S-2000).
  • the secondary antibody is Biotin labeled anti-mouse antibody (from Vector Labs # BA2000) dUuted 1:2000 in pre-filtered TPBS containing 1 % horse serum. The serum is then washed as already described, this time to remove the secondary antibody. Fluorescent cetection is performed using fluorescein anti-biotin reagent (Vector Labs # SP3040) prepared in TPBS and using the same 5 minute incubation described above. The specimen is then perfusion washed with de-ionized water to stop the incubation.
  • the specimen is removed from the Sweeny filter assembly and mounted with aqueous mounting medium suitable for use in fluorescence microscopy (Vector Labs VectaShield reagent # H-100), a cover sUp is placed over the specimen, and it is viewed microscopicaUy, using the same wavelength and filters conventionaUy used for fluorescent imaging.
  • aqueous mounting medium suitable for use in fluorescence microscopy Vector Labs VectaShield reagent # H-100
  • a cover sUp is placed over the specimen, and it is viewed microscopicaUy, using the same wavelength and filters conventionaUy used for fluorescent imaging.
  • 0.5 ml of alkaUne phosphatase labeled anti-mouse antibody (Vector Labs, Cat#AP-2000) diluted 1:2000 in pre-filtered TPBS containing 1.0% horse serum.
  • cplorimetric detection may be preformed using alkaUne phosphatase substrate incubation (with Vector Labs AlkaUne Phosphatase Substrate Kit, Cat#SK5200 prepared in 0.1M TRIS solution, at pH 9.5, as instructed by the manufacturer), and using the same 5 minute incubation as described for the primary antibody. Washing solution (deionized water) may be perfused through the specimen in the Sweeny filter to stop color development.
  • the specimen after being removed from the Sweeny filter assembly, may be rendered optically clear by dipping the composite film in xylene several times.
  • the sample may be prepared for microscopic viewing by placing a drop of mounting medium such as Accumount (Baxter Scientific) on to a glass microscope slide.
  • the composite filter is placed on top of the drop of medium, with the tissue specimen face up, and 2-3 drops more of medium are applied on the specimen surface and overlaid with a glass cover sUp.
  • the finished specimen may be viewed microscopicaUy for the presence of brown color stain in the nucleus, which mdicates the presence of PCNA protein.
  • a block of formaUn fixed paraffin embedded with human tonsU is mounted on a microtome chuck, and 4 ⁇ thick sections of tissue are made.
  • the tissue specimens are floated on the surface of a water bath, and each thin section is placed on a separate composite film.
  • the tissue specimen is affixed to the nitrocellulose surface of the composite film by microwave heating for 1 minute, or baking at 60°C for one hour.
  • the tissue is de-paraffinized by mcubation in xylene, and descending concentrations of isopropyl alcohols, using the foUowing sequence: xylene, xylene, xylene, followed by sequential isopropyl alcohol treatments for 3 minutes at each of the foUowing concentrations: 100%, 100%, 100% , 95%, 90%, 80% , 70%, and then water.
  • the distribution of water in the specimen is normaUzed by. soaking the specimen for one minute in a 75 % tertiary butyl alcohol (TBA) excipient.
  • TSA tertiary butyl alcohol
  • a disclosed embodiment of the excipient is 75 % TBA, 5 % sodium lauryl sulfate (SLS), 1 % polyvinyl alcohol (PVA), 1 % polyethylene glycol (PEG), 10% gelatin, and 1 % Tween-20.
  • the composite films (with the attached tissue specimens) are removed from the normalizing solution and excess solution is drained on to paper towels.
  • the specimen is frozen by placing the composite film on the platen of the lyophilization device (FIG. 9), in which the platen surface temperature has been pre-cooled to -45 °C (the condenser surface is cooled to below - 60°C).
  • Composite films with attached tissue specimens are inserted on different platens until aU the avaUable freezing platens are occupied.
  • the lid is closed, and a gasket pressed tightly against the opening to form a good seal when vacuum is applied.
  • a vacuum of 100 milUtorr is appUed in the lyophilization chamber.
  • the tonsU specimens are lyophUized at -45 °C for 3 minutes. Then whUe maintainmg a continuous vacuum of 100 mUUtorr in the lyophilization chamber, the temperature of the sample platens is increased to
  • Samples are prepared for perfusion of reagents by peeting or cutting the composite film (nitroceUulose and Nytran) from the support, and trimming to 13 mm.
  • the composite films are placed in the Sweeny filter and perfused as described in Example 4 with the blocking solution, primary antibody, washing solution, secondary antibody, washing solution, and colorirnetric reagent. Dehydration and preparation for microscopic viewing are performed as in Example 4.
  • Fluorescent antibody detection can be performed by deUvering antibody as described above, but where the antibody is Biotin Labeled Anti-mouse antibody (Vector Labs #BA2000) dUuted 1:200 in pre-filtered TPBS containing 1 % horse serum. The specimen is then washed as already described, this time to remove the secondary antibody. Fluorescent detection is performed using fluorescein-anti-biotin reagent (Vector Labs # SP3040) prepared In TPBS and using the same 5 minute incubation as described. Specimen is then perfusion washed with de-ionized water to stop incubation.
  • the specimen is removed from the Sweeny filter assembly and mounted with aqueous mounting medium suitable for use in fluorescence microscopy (Vector Labs, VectaShield reagent # H-1000), cover-sUpped and viewed microscopicaUy at the same length and filters conventionally used for fluorescent imaging.
  • aqueous mounting medium suitable for use in fluorescence microscopy (Vector Labs, VectaShield reagent # H-1000), cover-sUpped and viewed microscopicaUy at the same length and filters conventionally used for fluorescent imaging.
  • FIGS. 2-5 iUustrate key physical properties of the composite film 31, which are also summarized in Table 1.
  • FIG. 2 shows a composite film with a lyophilized cryostat breast cancer specimen shown after a Her-2/neu immunoassay.
  • FIG. 3 is a cross-section scanning electron micrograph of the composite film with the tissue specimen affixed to the nitroceUulose surface. NitroceUulose(NC)/Nytran(NY) composites were 185 ⁇ thick, with the exposed nitroceUulose surface layer comprising approximately 5 ⁇ . The nitrocellulose polymer penetrated the superficial nylon latticework (FIG.
  • the nitrocellulose surface of the composite formed a smooth contact plane for biological specimens, as shown by the intimate contact between tissue specimen (T) and film in FIG. 3.
  • FIGS. 4 and 5 contrast the surface of a NytranTM filter and a composite film having a nitroceUulose surface.
  • the Nytran surface (FIG. 4) comprises an "open meshwork" structure without discrete pores, whUe the working nitroceUulose surface (FIG. 5) of the composite film is planar with discrete round pores comprising approximately 50% of surface area (3.6 X 10 8 pores/cm 2 ).
  • the range in pore size on the surface of the nitroceUulose films was 0.01-0.8 ⁇ . Approximately 91 % of pores were 0.2-0.4 ⁇ in diameter (Table 1); 10% were 0.4-0.8 ⁇ , and 10% were ⁇ Q.2 ⁇ .
  • ⁇ P pressure (dynes / cm 2 )
  • A surface area (cm 2 ).
  • Table 1 also shows the permeabUity of laminate composite films to water under positive pressure.
  • the rate of water flow through laminate films was approximately 2 ml/min cm 2 , an average of 19 times slower than through Nucleopore films of the same surface porosity, with etched pores of the same diameter (0.2 ⁇ -0.4 ⁇ ).
  • the rate of water flow through composite films was approximately 10 times slower tiia ⁇ through the nylon support filter alone (Table 1), suggesting that the nitrocellulose thin film component of the composite determined permeabUity of the composite.
  • PermeabiUty of composite films was within the range estabUshed for Nucleopore standards of the same pore size and porosity (Table 1). However, since the 10 ⁇ thick nitrocellulose component of the 185 ⁇ thick composite film actuaUy determined flow rate through the entire composite, we could calculate from the Darcy equation, which relates thickness to flow rate and permeabUity, that the cast nitrocellulose film was actually 15 to 55 times less permeable (more tortuous) than the etched Nucleopore films of the same thickness with regular cyUndrical pores.
  • the porosity of tissue was sensitive to the temperature at which thaw-mounted cryostat specimens were re-frozen for lyophUization.
  • Mean pore diameter increased about 4 times, from an average of 0.392 ⁇ to an average of 1.6 ⁇ , as freezing temperature was raised from -70°C to -30°C, suggesting that pore size was a function of the re-freezing rate of ice in the specimen which determined the size of ice crystals.
  • the percent of tissue surface which comprised pores also increased with pore size and re-freezing temperature, from a minimum of 30% at -70° C to a maximum of 65% at - 30°C.
  • the distribution of pores was relatively homogeneous within specimens re-frozen at -30°C and -40°C, whereas at -70°C, there were areas of specimen with no visible pores.
  • Specimens re- frozen at -20°C were relatively poreless, with clusters of a few large vacuole-Uke pores, and appeared more "congealed," suggesting that the tissue may have collapsed due to formation of excessively large pores and involvement of too much of the tissue matrix.
  • 1 NF tissue not refrozen Tissue pore diameter and porosity were measured from scanning electron micrographs. Mean and S.D. in pore diameter are given for three separate tumors (2 specimens/tumor). Porosity is expressed as a range in % of specimen surface involved and where statistically meaningful (p ⁇ 0.05), pore density for the same specimens.
  • Pores formed by lyophitization at freezing temperatures of -70°C and -40°C were not readily visible in specimens observed at 400 X after chemical fixation and staining. Furthermore, neither the presence of pores in the range of 1 ⁇ (diameter) or less, nor the process of forming those pores, created anatomical artifacts that impaired cytology.
  • Major cell types could readily be differentiated, including glandular epitheUal, myoepithehal, endotheUal and lymphoid in lyophUized breast cancer tissue re-frozen at -70°C and -40°C.
  • Major breast cancer growth patterns could also be differentiated, including papUlary, cribiform and comedo.
  • Mitotic figures were readUy discernable in lyophUized specimens re-frozen at -70 °C and -40° C. At higher freezing temperatures (-30°C and -20°C), vacuoles were readily visible in fixed and stained specimens examined at 250 X. Cell matrices and mitotic figures were indistinct at these freezing temperatures. Spaces between cells were observed in specimens whether frozen or not. These spaces were artifacts related to the stretching of film during dehydration and not to the formation of pores.
  • pore sizes less than 1 ⁇ were not Ukely visible, and did not change contrast artifacts associated with resolution of ceUular structures in stained specimens. Ice crystal and resultant pore size is inversely related to the rate at which ice re-freezes, hence lower temperatures provide larger diameter pores whUe higher temperatures provide smaller pores. Surface pore diameter and internal pore diameter were virtually identical at a given freezing temperature. At a re-freezing temperature of -40 °C, pore diameter was nearly 1 u, and variation in pore size was only 31 % in multiple areas of three different breast tumors. The internal porosity of these specimens was vertically directional, giving the appearance of vertical "microchannels".
  • detergent-impregnated Film can contribute to the stabUity of microchannelled tissue.
  • crystalhne (microchanneUed) specimens have increased fragUity.
  • the detergent such as SDS or SLS
  • SDS or SLS can "weld" the specimen to a mtroceUulose film surface with an exceptionally high surface area for attachment.
  • the perfusion technology of the present invention can substantiaUy increase the efficiency of cytochemical assays.
  • a typical colorirnetric immunoassay comprises 8 basic steps involving 4-5 hours (retrieval 15-30 minutes; blocking, 30-60 minutes; primary antibody, 60-90 minutes; wash, 15 minutes; secondary antibody/linker/enzyme, 30-60 minutes; wash 15 minutes; substrate, 30 minutes; wash, 15 minutes).
  • In situ hybridization typically comprises even more steps and longer incubations. However, by percolating reactants into porous tissue under pressure, we anticipate blocking and probe incubations will be completed within one minute. This savings in time wiU be matched by savings in reagent cost, because much smaller volumes of reagents need to be used in the more efficient perfusion assays.
  • Perfusion assays can be performed with microliter volumes of expensive reagents, instead of the liters of reagents that are used in the prior art.
  • the composite films 31 are manufactured by a process of placing a clean-release adhesive liner over a depth (or other) filter to expose a circular filter area, using the lmer to mask the filter and adhere it to a glass microscope sUde. Affixing the composite film to a microscope slide facUitates tissue mounting and heat transfer during freezing and drying, and the film can be easily removed from the slide for filtering. Composite films are polymer cast onto the exposed filter area. After a quench process to form pores, a detergent was added and dried on the Film.
  • nitrocellulose films can be cast on a variety of microporous support materials, which are shown in Table 3.
  • Nytran was preferred because it has a 0.2 ⁇ pore size (pore size in these depth filters is a measure of particle exclusion limit rather than surface or internal pore size), an exceptionally smooth surface, tough pUabUity, absence of a fibrous core, and chemical properties that promoted nitroceUulose bonding without dissolving in the acetone solvent or pore formers.
  • the 0.2 ⁇ pore size was selected over 0.45 ⁇ , because that smaUer pore size provides an optimally smooth surface for polymer casting, whUe sttil preserving adequate flow characteristics.
  • the Nytran that is chosen preferably has light transmittance of at least 60% of glass (i.e. the percent of visible and UV tight that is transmitted though the Nytran should be at least 60% of the same light that would be transmitted through glass of the same thickness).
  • Maximum Strength Nytran (also without internal fiber support), can be equivalent to the Nytran in aU properties shown in Table 3 (including light transmittance). Neither product autofluoresced in ultraviolet light. However, the refractive indices of nylon and nitroceUulose should ideally be identical.
  • Another potential support material is nylon stretched 10% of its length in isopropanol, but this support allows the nitroceUulose film to stretch, which can cause break artifacts in the specimen matrix.
  • porous support materials include porous glass coverslips (0.5 pore size from Corning/Kamperman); etched ceramic filters (0.2 ⁇ pore size from Whatman, Anopore); and regenerated cellulose (0.2 ⁇ pore size from Schleicher & Schuell).
  • the glass, ceramic and regenerated cellulose materials have suitable visible/UV tight transmittance and stretch-resistance.
  • a silane can be used to covalently bond the nitrocellulose to the glass.
  • a suitable poUshed glass material which is 200 ⁇ thick (the approximate thickness of a #1 cover stip), with pores approximately 0.5 ⁇ in diameter, is avaUable from Corning (Vycor
  • the porous support material preferably has at least 60% hght transmittance, no autofluorescence in ultraviolet hght, 100% focal plane uniformity, less than 1 % Unear distortion in 100% isopropanol, and 100% cytologic resolution.
  • Lyophilization Parameters Lyophilization of the tissue specimen can be performed under a variety of conditions, with the goal of providing a porous specimen with vertical microchannels through which a liquid can be perfused. This goal can be achieved by a device (such as that shown in FIG. 9) which freezes the specimens a rate greater than 5°C/sec (for example 20°C/sec to 100°C/sec or more), under weU regulated vacuum and temperature control.
  • a 1: 1 ratio between drying surface area and condenser surface area is adequate for optimum condenser efficiency.
  • composite films were soaked in an excipient containing 75% tertiary butyl alcohol (TBA), 5 % sodium lauryl sulfate (SLS), 1 % polyvinyl alcohol (PVA), 1 % polyethylene glycol (PEG), 10% gelatin, and 1 % Tween-20.
  • TSA tertiary butyl alcohol
  • SLS sodium lauryl sulfate
  • PVA polyvinyl alcohol
  • PEG polyethylene glycol
  • Tween-20 1 % Tween-20
  • the composite films may also be soaked in a detergent such as SLS or SDS, which was then dried on the surfaces of the composite films.
  • the detergent plasticized the film, welded the specimen to film, extracted weakly (non- covalently) bound membrane proteins, and enhanced vertical water/solute transfer to film through capUlary action.
  • the detergent also apparently Unproved the uniformity of solute water distribution (ice nucleation sites) within specimen.
  • the detergent is also beheve
  • excipient such as 75 % t-butyl-alcohol
  • tissue specimen prior to lyophUization also helps improve uniform distribution of water in the specimen, enhances nucleation of ice, normalizes the eutectic temperature and refreezing rate of ice in specimens that are otherwise intrinsicaUy heterogeneous with respect to these properties, and helps preserve the structural stabUity of porous specimens and labUe analytes in the dried state.
  • the excipient is beUeved to normalize the eutectic temperature (to about -38°C) by imposing its own water content and solvent distribution on the specimen, which is greater than the inherent water concentration of the specimen.
  • the hybridization assay we employ is essentiaUy that of Lawrence and Singer, Nucleic Acids Res. 13: 1777-1799, 1985, using a full length cDNA and hybridization at 42°C in 50% formamide, except that digoxygenin-labeled cDNA is employed and hybridization is detected using FITC -labeled anti-digoxygenin antibody.
  • the composite film assay format also differs from the glass slide format in that the protease digestion step is omitted as unnecessary for lyophiUzed specimens. Data are obtained using a Bio-Rad Image Analyzer, and expressed as % reactive ceUs (the target is diffusely cytoplasmic at 250-400 X), with variation determined using a Factorial Anova with nested components.
  • the intensity of a colorirnetric signal can be quantitated, in accordance with known methods.
  • a photomicrograph of the specimen can be obtained, and the images digitized.
  • a pixel intensity scan is taken along a computer generated line between markers on the digital image, and the intensity of pixels determined along that line.
  • the maximum pixel intensity is an indication of the concentration of the probe (such as a monoclonal antibody) that has hybridized to the tissue.
  • the intensity of this signal can in turn be correlated with the concentration of the protein of interest in the tissue.
  • Pathology specimens that are suitable for immunohistochemical analysis include ceUs that express labile proteins and mRNA markers (for example Her-2, PgR, EMA, A blood group, and b- actin), from any type of animal or plant tissue.
  • the specimen thickness may vary widely, for example from 1 ⁇ to 100 ⁇ (although specimens may be outside this and other ranges given in the specification).
  • the thickness of the composite film can also have a variety of range of thicknesses, as long as the thickness does not interfere with imaging and stain backgrounds.
  • a thickness of less than 200 ⁇ (for example 185 ⁇ or less) is an example of a suitable thickness.
  • Perfusion flow rates can also vary widely, but are optimally as fast as possible without compromising the specimen structure, for example 0.5 ml/min/cm 2 to 5 ml/min/cm 2 .
  • Perfusion pressure is optimaUy less than 30 psi.
  • a range of exemplary lyophiUzation temperatures is -20°C to -70°C, at a lyophUization pressure of 200 millitorr to 70 miUitorr.

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Abstract

Selon la présente invention, on dispose un spécimen tissulaire (32) sur un support composite (20) de nitrocellulose (30) et de nylon poreux (19), et on le lyophilise de façon à produire dans le spécimen des microcanaux verticaux. On fait alors perfuser par le spécimen poreux (32) et le support (20) des réactifs immunocytologiques, ce qui assure un contact intime entre le spécimen (32) et les réactifs.
PCT/US1997/019871 1996-11-01 1997-10-31 Cytoperfusion de specimens tissulaires WO1998020353A1 (fr)

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EP1174702A1 (fr) * 2000-07-20 2002-01-23 Medic SRL Dispositif automatique pour le traitement des lames de microscope pour la coloration des spécimens biologiques
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WO2003065014A1 (fr) * 2002-01-30 2003-08-07 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Supports d'echantillons pour la cryoconservation d'echantillons biologiques
US6673620B1 (en) 1999-04-20 2004-01-06 Cytologix Corporation Fluid exchange in a chamber on a microscope slide
US6921637B2 (en) 2000-05-04 2005-07-26 The Cbr Institute For Biomedical Research, Inc. Colloid compositions for solid phase biomolecular analytical, preparative and identification systems
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US7214477B1 (en) 1999-07-26 2007-05-08 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Layered device with capture regions for cellular analysis
US7297497B2 (en) 2002-08-16 2007-11-20 Decision Biomarkers, Inc. Substrates for isolating, reacting and microscopically analyzing materials
US7838222B2 (en) 1999-07-26 2010-11-23 United States of America/ NIH Methods, devices and kits for multiplex blotting of biological samples from multi-well plates
WO2011067221A1 (fr) * 2009-12-02 2011-06-09 Whatman International Ltd Méthodes et systèmes de traitement d'échantillons sur des substrats poreux
US8283158B2 (en) 2002-11-25 2012-10-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method and apparatus for performing multiple simultaneous manipulations of biomolecules in a two dimensional array
WO2014093206A1 (fr) * 2012-12-13 2014-06-19 Merck Sharp & Dohme Corp. Granulés sphériques lyophilisés d'anticorps anti-il-23

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US7214477B1 (en) 1999-07-26 2007-05-08 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Layered device with capture regions for cellular analysis
US6602661B1 (en) 1999-07-26 2003-08-05 20/20 Genesystems, Inc. Methods and arrays for detecting biomolecules
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US6921637B2 (en) 2000-05-04 2005-07-26 The Cbr Institute For Biomedical Research, Inc. Colloid compositions for solid phase biomolecular analytical, preparative and identification systems
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WO2003065014A1 (fr) * 2002-01-30 2003-08-07 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Supports d'echantillons pour la cryoconservation d'echantillons biologiques
US7435582B2 (en) 2002-01-30 2008-10-14 Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Sample carrier for cryoconservation of biological samples
US7297497B2 (en) 2002-08-16 2007-11-20 Decision Biomarkers, Inc. Substrates for isolating, reacting and microscopically analyzing materials
US7384742B2 (en) 2002-08-16 2008-06-10 Decision Biomarkers, Inc. Substrates for isolating reacting and microscopically analyzing materials
US8283158B2 (en) 2002-11-25 2012-10-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method and apparatus for performing multiple simultaneous manipulations of biomolecules in a two dimensional array
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CN102741697A (zh) * 2009-12-02 2012-10-17 沃特曼国际有限公司 用于处理多孔基片上的样品的方法和系统
JP2013513097A (ja) * 2009-12-02 2013-04-18 ワットマン・インターナショナル・リミテッド 多孔質基材上で試料を処理する方法及びシステム
US8685749B2 (en) 2009-12-02 2014-04-01 Whatman International Limited Methods and systems for processing samples on porous substrates
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WO2011067221A1 (fr) * 2009-12-02 2011-06-09 Whatman International Ltd Méthodes et systèmes de traitement d'échantillons sur des substrats poreux
US9254484B2 (en) 2009-12-02 2016-02-09 Whatman International Limited Methods and systems for processing samples on porous substrates
US10365190B2 (en) 2009-12-02 2019-07-30 Whatman International Limited Methods and systems for processing samples on porous substrates
WO2014093206A1 (fr) * 2012-12-13 2014-06-19 Merck Sharp & Dohme Corp. Granulés sphériques lyophilisés d'anticorps anti-il-23

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