histokinetic by dr narmada
TRANSCRIPT
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 metal container or in a plastic bag in 10% formalin. If formalin is not available at hand, place the specimen in refrigerator 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
SpecimenHistology lab
Registration
Fixation
Grossing
Labelling
Dehydration
Clearing
Infiltration & impregnation
Embedding Trimming
Section cutting
Deparaffinization
Hydration
Staining
Dehydration
Clearing
Mounting
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
Decalcification
A 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 type 2- Moving fluid type.
Histokenette Automatic Tissue Processor
Tissue Cassettes
Tissue Processor With Vacuum And Fume Control
Vacuum Tissue Processor
Linear Tissue Processor With Touch Screen
PROCESSING SCHEDULEAutomated processing schedule1-Overnight schedule2-short processing schedule
Manual processing schedule
Over night schedule1- 10% formaline.2- 70% alcohol3- 95% alcohol4- 100% alcohol5- 100% alcohol6- 100% alcohol7- 100% alcohol8- 100% alcohol 9- xylene10- xylene11- wax12- wax
0 hrs½ hrs.½ hrs½ hrs1 hrs1 hrs1 hrs½ hrs1 hrs2 hrs2 x1/2 hrs4 hrs
Schedule for processing for small biopsy or for urgent work1- 10% formaline.2- 95% alcohol3- 95% alcohol4- 100% alcohol5- 100% alcohol6- 100% alcohol7- 100% alcohol8- 100%
alcohol/ethylene9- xylene10- xylene11- wax12- wax
20 min vacuum heat5 min y
450c5 min5 min5 min5 min5 min5 min5 min5 min5 min5 min
Rapid technique for thin slices of tissue
1- 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
Manual processing schedule for blockes
1- 70% alcohol2- 95% alcohol3- 95% alcohol4- 100% alcohol5- 100% alcohol6- 100% alcohol7- 100% alcohol8- xylene9- xylene10- wax with vacuume11- wax with vacuume12- wax with vacuume
0900 hrs to 1000 hrs1000 hrs to 1100 hrs1100 hrs to 1300 hrs1300 hrs to 1430 hrs1430 to 1600 hrs1600 to 1730 hrsovernight0900 to 1000 hrs 1000 hrs to 1130 hrs1130 hrs to 1230 hrs 1230 hrs to 1400 hrs1400hrs to 1600 hrs
½ hrs½ hrs1 hrs1 hrs1 hrs½ hrs1 hrs2 hrs2 x1/2 hrs4 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.
DehydrationDehydrating agents 1-Alcohols- ethenol, methanol,
isopropanol,Polyethylene glycols (PEG) , diaxone
2-Other dehydrantsAcetone , Tetrahydrofuran , 2,2
dimethoxypropane Phenol,
Clearing
1-. It is the process in which water from cell & tissues is removed & is replaced by a fluid in which wax is soluble
2- .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 & Impregnation
Infiltration – 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 Feature
Commonly used
54 c
Hard wax 60 c Hard fibrous tissue
Soft 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
Used 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.
MICROTOME
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)
STAININGDeparaffinized (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 min
Counterstain (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 study
Essential 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 their natural position .
it is practically impossible to obtain and correlate serial section
Methods Freezing 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