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It is
obvious that for the pathologist to recognize accurately a
morphologic pattern must have access to the best preserved
tissues and histologic sections to optimize the resolving
power of the light microscope (0.2 um). This is not possible
with the paraffin sections. In our Laboratories we have
replaced paraffin embedding with epoxy embedding. This
method (High Resolution Light Microscopy or HRLM) permits
full utilization of the resolving power of the light
microscope and needs no routine special stains. For the
complete, comprehensive examination of the renal biopsies we
need only two samples: one for HRLM-TEM studies and one
sample for IH studies. "THE SAMPLES MUST NOT BE MINCED." The
tissue for HRLM-TEM is fixed in aldehydes and post-fixed in
osmium tetra oxide. Embedding is accomplished with Spurr
epoxy, stat (3h) or overnight schedules. The blocks with the
intact samples, are sectioned in an ultramicrotome with
glass knives, stained with toluidine blue and basic fuchsine
(TB-BF). Serial sections are studied under the light
microscope obtaining resolution of the smallest lesions
possible in the light microscope (02.um, about the size of a
mitochondria) without the need of special stains. Probably
more than 90% of the diagnostic features are visible in
these sections for HRLM ( ). STEP
BY STEP DIRECTIONS: The specimen is submitted in fixative of
choice, our laboratory strongly recommends and, upon
request, provide Paraformaldehyde-G Fixative for High
Resolution Light Microscopy (HRLM) - Transmission Electron
Microscopy (TEM). The documentation of what fixative (upon
doing the gross description and on the PROCESSING SCHEDULE)
the specimen is submitted is very important as the final
HRLM-TEM stain will be affected.If processing using the EM
automatic processor, program # 1 would be used. When
manually processing, the tissues ARE NOT removed from the
original vial they are submitted in.When any of the reagents
used to process are introduced to the specimen the vial is
capped and AGITATION is extremely critical, especially with
this STAT technique. 1.POST-FIXATION begins with Working Osmium Tetroxide. Working Osmium Tetroxide is added to the specimen (the volume has to be 1 to 10) in the vial. The vial(s) are placed inside an Explosive Proof Refrigerator for 30 minutes. 2.Remove the specimen vials from the Explosive Proof Refrigerator and working with a vent -a -hood collect and discard the working osmium tetroxide in Hazardous Waste Container DO NOT POUR DOWN THE DRAIN, CHECK WITH YOUR SAFETY HAZARDOUS WASTE MANAGEMENT IN YOUR INSTITUTION. And now dehydration begins with 25% Ethanol. Dehydration is done at room temperature, utilizing a vent-a -hood. REMEMBER, AGITATE 3.DEHYDRATION begins with three changes of 25% Ethanol to total 1-2 minutes. 4.Discard the 25% Ethanol and add 50% Ethanol, agitate for 1 minute. 5.Discard 50% Ethanol and add 75% Ethanol, agitate for 1 minute. 6.Discard 75% Ethanol and add 90% Ethanol, agitate for 1 minute. 7.Discard 90% Ethanol and add Absolute Ethanol for three changes to total 3 minutes-agitate. 8.Discard the last change of Absolute Ethanol and add EM Grade Acetone for two changes to total 3 minutes-agitate. 9.Discard the last change of Acetone and begin infiltration with a 1:1 mixture of EM Grade Acetone and prepared Firm Epoxy Media. INFILTRATION IS ENHANCED IN A 40oC incubator 10.INFILTRATION begins with a one to one mixture of EM Grade Acetone and prepared Firm Epoxy Media for two changes to total 20 minutes, remember to cap, agitate and place in 40o incubator. 11.Discard the last 1:1 mixture and add prepared firm epoxy media for six changes of 10 minutes each to total 1 hr, remember to cap, agitate and place in 40o incubator. 12.After the last change of prepared Firm Epoxy Media, the tissues are now ready to be embedded. For STAT cases, the specimen is embedded in the BOJAK mold using Hard Epoxy Media and microwaved at Control 50 for 5 minutes at clock position 9, 12, and 3. This will total 15 minutes. For next day cases, the specimen is embedded in the BEEM capsules using Hard Epoxy Media and polymerized in a 70oC incubator. 13.EMBED the tissues in BEEM capsule only after appropriately labeling the inside of the BEEM capsule with the Referred Surgical number that has either been written in india ink or typed on bonded paper. Fill the BEEM capsule with prepared epoxy media of choice. 14.Place the tissue that corresponds to the referred surgical number inside the BEEM capsule and let gravity pull the tissue towards the bottom . Ori '¨ê $Ä ( ðnecessary wh '© rying to align needle biopsies next to each other. 15.POLYMERIZE by placing BEEM capsules in a 90oC incubator for 1hour and 45minutes. 16.Remove the BEEM capsule holder from the 90oC incubator and place BEEM HOLDER and epoxy blocks, inside the lower part of a 15oC refrigerator for 10 minutes. Remove the BEEM capules from the refrigerator and thenplace them on the BEEM remover and pop out the epoxy blocks. Trim the excess epoxy, without touching the tissue using an oil free straight edge razor. DO NOT USE THE INFRARED LAMP TO SOFTEN EPOXY BLOCKS, THEY WILL SHATTER! Blocks are now ready for sectioning and staining as per routine. Beem capsules that have been polymerized overnight are removed from the 70oC oven and the polymerized epoxy blocks are removed from the BEEM capsule using the BEEM remover. The blocks polymerized in the 70oC incubator are trimmed of the excess epoxy, without touching the tissue, with an oil free, straight edge razor. |
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REFERENCES / REFERENCIAS Bancroft, J.D. and Stevens, A.: Theory and Practice of Histological Techniques, Churchill Livingstone. third Ed., 1990. Flores TR, Hoffmann EO and Coover J: Improved High Resolution Light Microscopy and Transmission Electron Microscopy Techniques in Diagnostic Pathology. J Histotec:20 (#1): 45-52, March 1997. (Click left for complete article). Flores TR, Hoffmann EO and Velasquez M: Stat high resolution light microscopy (HRLM) - Transmission Electron Microscopy (TEM) Techniques for transplant renal biopsies. J. Histortec 21 (#3): 213-217, 1998. (Click left for complete article). Glauert, A.M.: Fixation, Dehydration and Embedding of Biological Specimens. North Holland Publishing Co., New York, 1975. Hoffmann E.O., Coover J, Flores TR, et al: Improvements for routine renal pathology. Lab Med 16: 237, 1985. Holliday MA, Barratt TM, Avner ED (1994): Pediatric Nephrology, 3rd edn. Williams and Wilkins, Baltimore. McDowell, E.M., and Trump, B.F.: Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Path Lab Med 100:405, 1976. |
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Results of the stain |
Resultados de las coloraciones |
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Alcohol hyaline: Pink with purple core Amyloid: Light blue, pink Bacteria: Blue or red Basophil granules: Dark blue Bile granules: Light blue Bowman's capsule: Light blue- pink Cells, necrotic: Dark blue Collagen, cartilage (Type III): Red Collagen, interstitial (Type I, II): Red (Extra-cellular) Collagen, basement membrane (Type IV): Light blue to pink Cytoplasmic sap: Pink Desmosomes: Dark blue Effaced podocytes: Blue line Elastic tissue: Purple Eosinophil granules: Dark blue Erithrocytes: Blue Fibrin: Dark blue Fungi: Blue, red Glycogen: Pink, red (intracellular) Hyaline material:(Hyalinosis) Blue-gray Immuno-protein deposits: Dark blue Laminated bodies (Myelinosomes): Dark blue Lipid droplets: Green to blue Lipofushsin: Dark blue and green Lysosomes: Blue, red or gray Melanin granules: Dark brown, blue Microbodies: Blue Mucus: Blue, pink or red Myelin: Dark blue Neutrophil granules: Light blue Nuclear envelope: Dark blue Nucleolus: Dark blue Nucleus: Light blue to pink Phagolysosomes: Blue to pink Proteinaceous deposits: Dark Blue Red cells: Dark Blue Secretory granules: Blue or pink Tonofilaments: Dark blue Viral inclusions: Blue or pink |
Amiloide: Azul claro, rosado. Bacterias: Azul o rojo Cápsula de Bowman: Azul claro-rosado Células necróticas: Azul denso Citoplasma: Azul claro-rosado Colageno intersticial: Rojo (Extracelular) Complejos inmunes: Azul denso Desmosomas: Azul obscuro Eritrocitos: Azul Fagolisosomas: Multicolor Fibras elasticas: Rojo-purpura Fibrina: Azul obscuro Granulos de melanina: Cafe obscuro-azul Granulos de mucina: Rojo-azul Glicogeno: Rojo (Intracelular) Gotas de lipidos: Azul-verde Gránulos de los basofilos: Azul Gránulos de los eosinofilos: Azul Gránulos de los neutrofilos: Azul Gránulos mucina: rosado Globulos rojos: Azul Hialino alcoholico: Rosado-purpura Hialinosis: Azul-gris Hongos: Azul, rojo Inclusiones virales: Azul-rosado Inmunoproteins: Azul obscuro Lipofucsina: Azul-verde Lipidos: Azul-verde Lisosomas: Azul, rojo, gris Material hialino (Hialinosis): Azul claro-rosado Membranas basales: Azul claro-rosado Mielinosomas: Azul obscuro Mitocondrias: Azul-rosado Microcuerpos: Azul Nucleo: Azul claro o rosado Nucleolo: Azul obscuro Proteinas: Azul Polisacáridos: rojo Parasitos: Azul-rosado Podocitos, borramiento: Linea azul Sobre (membrana) nuclear: Azul Tonofilamentos: Azul Virus (Inclusiones): Azul o rosado
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