As the name suggests in moving tissue type processors, the tissue is moved from reagent to reagent, on the other hand in moving fluid type, the tissue is kept stationary and different reagents are passed through the tissue one by one using vacuum. Infiltration under vacuum is more effective and produces superior samples.
Tissue processor: construction: all exterior parts are powder coated in RAL 9002 12 stations, 10 glass containers, 2 stainless steel containers, 1 transport basket is included, for approx. 110 cassettes, optional twin basket for approx. 220 cassettes lifting mechanism for 1 transport basket rear connection possibility for hose (100 mm diameter), with fan or active carbon filter as option electronical controls with multi power supply 85-264 V. Worldwide operation without power adjustment display and operating panel with programming keys in case of power failure the basket is moved in a programmable
Histokinetic by dr narmada
It is processing and preparation of the tissue of body in such a manner as to satisfactory study of the tissues can be done.
Handling of SpecimenSpecimen should be transported in glass, plastic or metalcontainer or in a plastic bag in 10% formalin.If formalin is not available at hand, place the specimen inrefrigerator at 4oC to slow down autolysis.fresh material is needed for the following purpose:1. Frozen section2. Immunocytochemistry3. Cytological examination4. Microbiological sampling before histopathology5. Chromosome analysis6. Research purpose7. Museum display
Basic stepsPreparation of the tissuesProcessing of the tissuesPreparation of the sectionsStainingMounting
Fixation - AIMPrevent putrefaction & autolysisPreservation of cells & tissue constituentsHardening of soft tissuesConversion of semifluid consistency of cell to an irreversible semisolid consistencyAlteration of refractive indices to varying degree which enables unstained components to be seen easily.
Ideal fixativesCheap & easily availableStable & easy to handleFix quicklyMinimal loss of tissueEven penetration
Reagents employed as fixativesFormaldehydeMercuric chloridePotassium dichromatePicric acidEthyl alcoholGlutaraldehydeOsmium tetraoxide
DecalcificationA process to remove calcium from bone and other mineralized hard tissue in order to facilitate the process of cutting thin section .Stages:-I. Selection of tissueII. FixationIII. DecalcificationIV. Acid neutralizationV. Washing
Processing of tissue Embed the tissue in a solid medium.Firm enough to support the tissue and enable thin sections to be cut.Soft enough not to damage the knife of tissue.
There are two kinds of tissue processors:1- Moving tissue type2- Moving fluid type.
Schedule for processing for small biopsy or for urgentwork1- 10% formaline. 20 min vacuum heat2- 95% alcohol 5 min y 450c3- 95% alcohol 5 min4- 100% alcohol 5 min5- 100% alcohol 5 min6- 100% alcohol 5 min7- 100% alcohol 5 min8- 100% 5 min alcohol/ethylene 5 min9- xylene 5 min10- xylene 5 min11- wax 5 min
Manual processing scheduleRapid technique for thin slices of tissue1- carnoys fluid- 45 min2- 100% alcohol x 6 15 min each3-xylene 10 min4- xylene 15 min5- wax 20 min6- wax 45 min
Manual processing schedule for blockes1- 70% alcohol 0900 hrs to 1000 hrs2- 95% alcohol 1000 hrs to 1100 hrs3- 95% alcohol 1100 hrs to 1300 hrs4- 100% alcohol 1300 hrs to 1430 hrs5- 100% alcohol 1430 to 1600 hrs6- 100% alcohol 1600 to 1730 hrs7- 100% alcohol overnight8- xylene 0900 to 1000 hrs9- xylene 1000 hrs to 1130 hrs10- wax with vacuume 1130 hrs to 1230 hrs11- wax with vacuume 1230 hrs to 1400 hrs12- wax with vacuume 1400hrs to 1600 hrs
Processing of tissueImportant steps of tissue processing by paraffin wax technique.a)Dehydration.b)Clearing.c) Infiltration.d)Embedding.
Factors influencing the rate of processing-a)Heat ( increase the rate of penetration, but limited to 45 degree)b)Agitation (tissue lies on the base of the container ,rate of exchange of fluid is much less)c) Viscosity ( quickness of impregnation due to lower viscosity of paraffin in fluid state )d)Vacuum ( little increase dehydration and clearing but reduces the impregnation time)
DehydrationI. Water is completely removed from the fixed tissues. II. Tissue blocks are placed in cassettes with the identification number. III . Passed through increasing concentration of alcohol with changes in each concentration.
Clearing 1-. It is the process in which water from cell & tissues is removed & is replaced by a fluid in which wax is soluble2- .Most commonly used agent is xylene.3-. Xylene is miscible in both paraffin wax & alcohol.4-. Replaces alcohol & make room for paraffin.5- Other clearing agent – toluene , benzene , chloroform & cedar wood oil.
Xylene or toluene or benzene :Advantages – Cheap and rapid in action, can be used for almost all tissue, used for both paraffin and celloidin embedding, benzene has less hardening effect then xylene.Disadvantages –a)Make the tissue brittle if kept in the fluid for a longer period.b)Excessive shrinkage for delicate tissue.c) May cause dermatitisd)Benzene is more inflammable and toxic and known to be carcinogenic.
Infiltration & ImpregnationInfiltration – xylene is eliminated from the tissue by diffusion in the surrounding melting wax.Impregnation – wax diffuses in the tissue by replacing the xylene.It maintain the intra cellular structure during the section cutting on microtome.
EmbeddingI.Casting or blocking.II. Infiltrated & impregnated tissue is places in warm liquid which forms a firm block after cooling.III. Enables the tissue to be cut on a microtome.IV. Most commonly used material is paraffin.V. Leuckhard embedding box ( consisting of two L shaped pieces of heavy metallic material brass) arranged on a glass plate.
Ideally an infiltrating and embedding medium should be:• soluble in processing fluids• suitable for sectioning and ribboning• molten between 30°C and 60°C• translucent or transparent; colorless• stable• homogeneous• capable of flattening after ribboning• non-toxic• odorless• easy to handle• inexpensive
Paraffin Melting point FeatureCommonly 54 cusedHard wax 60 c Hard fibrous tissueSoft wax 45 c Fetal & areolar tissue
Celloidin media ( nitrocellulose )1.Used when it is desired to avoid the use of heat in CNS2.It is rubbery material.3.Give better support to tissue like skin, sclera & subcutaneous tissues.Gelatin media – for friable tissues like lung.Resin , Agar
MICROTOMEUsed for cutting paraffin tissue sections of uniform thickness. A knob on the machine is used to adjust the thickness of section.A knife is fixed in a clamp . The tissue block is drawn across the knife edge, the top and bottom of the block should be parallel and horizontal and at least 1 mm of paraffin should be present on all side of tissue.Then ribbon of sections is transferred to warm water.
MICROTOMERotary microtome – for paraffin embedding.Rocking microtome – for paraffin embedding.Sliding microtome – for celloidin embeddingFreezing microtome – for frozen sectionCold ( cryostat) – for frozen sectionUltramicrotome – for electron microscopeLaser microtome-for contact free slicing
Trimming of the paraffin block.Attach the block to the microtome.Cutting of the section.Fix the section on the slides. adhesives – starch paste albumin ( glycerol + white egg + distilled water)
STAINING Deparaffinized (2 jars of xylene – each 2 min) 2 jars of alcohol – each for 2 min.Rehydrated (90% alcohol – 1min f/b 70% alcohol for 1 min) Rinsed in water Stained in harris haematoxylene for 2 – 5min Washing in running water till sections turn blue
Differentiation (1% acid alcoholic solution for 10 sec)Dip in 0.5% hydrochloric acid, nuclear aaper dark purple.( bluing) Rinse in water for 10-15 minCounterstain (1% aqueous solution of eosin for 1-3 min) Rinse in tap water Dehydrate Clearing in xylene Mounting ( DPX / Canada balsam)
Frozen sectionMethod of sectioning of tissue after making the tissue hard by rapid freezing.Advantages – rapidly prepared, Minimum shrinkage of tissue, Every method of staining is allowed especially suitable for immunochemistry and almost obligatory for enzyme studyEssential technique for demonstration of certain lipid and enzymes.
Disadvantages –sections are thick and sometime difficult to interpret properly,not always possible to maintain the structural element in theirnatural position .it is practically impossible to obtain and correlate serial section
MethodsFreezing microtome with carbon dioxide which is popular method.Freezing microtome with thermoelectric molecules.Use of refrigerated microtome ( cryostat).10 % formal saline is most common fixative used, fresh unfixed tissue may be used.Tissue thickness should not exceed 3 mm.
AdvancesUse of different type of resins as alternative to wax.Large block preparation and large sections for study of whole pathological region of tissue.Specimen radiography or thin section ultrasonography on freshly removed specimen to be sure that the whole pathology has been removed.Microwaves for quick fixation, processing.
Microwave processing of tissueTissue is cut on small pieces ---- tissue block are placed in isotonic saline and subjected to microwave irradiation for 120 sec. (full power )- ( 50 % power) dehydration by 70 % alchohal for 4 min, 100 % alchohal for 5 min, chloroform for 5 min then impregnation in wax for 5 min---- embedding- section cutting.
THANK YOU PRESENTED BY – Dr. Narmada Prasad Tiwari