Tissue Processing

Processing
Processing of the tissue involves rendering the tissuc frn So thet hn
Secuonscan be cut and studied under thc microscope. Thc procedures hvolved
procesSing the tissuc are Collection, Pixation, Dehydration, Clearing Ond
Enbedding.
Collection, labeling, and fixation of the specimens
(Whe specimens processcd in a histopathology laboratory arc thc tissue and
Organs removed from the living body and thc organs obtaincd from autopsy afier
:e death of the patient,)
Immediately after removalfronm the body, minute tissues as wellas large
specimens, should be fixed intL0% formalin or otier fixatives of choice wh:ch
should be about 20-25 times the volúnc of the tissue. Thc spccimens can bc
collected in plastic or glass containcrs. Soon afier collection, a labci containing
the name, age, sex, hospital number and ward of the patient should be pasted on
to the containers)This should be accompanicd by arequisition form, bearing the
essential details about the patient and thc spccimen.
As soon as the specimen reaches/the laboratory. the technician_shoule
verify the details written cn 1'c latelas wcllas the requisition form andaçcepti:
only if theinformation tallics}(Ore llhis is done, the specimcn should be given a
number, fÍr example inST25/84, Sacnotes the surgical spccincn 125, the number
of the specimen in scrial o:der, and 84,the ycar uf the reccipt of the specinen.
Thissurgical number alongwitlh the paticnt's nane, age, sex, hospital number,
wardand service shouid be cterud in a sepatc ster for rcfercnce.
 cim thickness nd ixd
(Large specimcLS are cut into sliccs of I to 2
3-6 hours of fix zt3a. 4
adequatc iomalin for 24 hours. S:nall tissues thick scctions areta' en fron u
require
adecquate fixation, )l to2cm: Square aid 41o5 mm
chs and plaCcdin tissue processingbaskets. These are periordiu
tissue is placed sen2cte i
conlaincrs having,one or four compart1ncnts. Each
pencil u strp
with its umbcr written with a graphite
smpieompartmcnt
whte papcr, Sallcr SDcCICs with their numbers are placed on a i t u
bD
odcd sccurcly, Miule specimcns can be stained with S0SIU
laCmp 0na filet papç1 for futurc idcntifictou.
Foilowiny fixation lhe excet fis:tive is teho:cd to ensure proper Miili
lixativCN Afctod of rmnal ol ex eSs fiaIne
Whjnwatcr fur i0-15nintcs ani!ac.
alolhol
Zenker o Jclly's Fuid Wish waier fu 2L24 hours
Bouin's Flud Waslh in S0%d 70"alcohol for 1i mim
Carnoy's Tluld T'ranslç1 decty o absol.taluboi.
Jehvdration
The.paralln io ich the tissuc is cnubeicted is not nscit
Hcnce, water an thc Ússac houid be r m07
This proccss IS Lalld duhydration.
Dehvdrating agents Idvantuges
Ethvl slcohol 1. Noi-toxic Disadvantaes
Lon: pDods it st sol
?. Misc1ble with water
3 No shinkaye of alcoho iny ans
daing, of the t:*
aluohol s M.
4 ldeallor de icatc
isopropyl alcool
of cthyalcol
 Acelone 1: Rapid dehyd1 ation 3
witlun 1. Requires a ciearing
4
1/2 -2 hours agent
2. Volume rust be 20
times IIore than the
tissue
3. Overhardens the tissue
if kept for long.
Diuxane 1. Botl: dehydrating 1, Expens1ve
and cieai gagent 2.-Toxic
2. Miscible with water, 3. Odurous
alcobol, xylene and
paraflin
3. Fast dehydrant
4. Less shrinkage than
ethyi alcohol
Technique of Dehydratior
Following fixation- aid washing the tissue is passed through a series of
graded alcohols or othe dehydrating agents. The total dehydration timi
depends on the volurne and type of tissue and the dehydrating agents.
Delyaration Time for alcohol
70% alcohol lhour
80% alcohol 1hour
95% alcohol 1hour
95% alcohol 1hour
Absolute alcohol-: Ihour
Absolute alcohol lbour
Absolute alcohol-3 1hour
1
2.
The volume of dehydrating agents should be adequate.
1oensure that the final bath of alcobol is free óf water, nhyarous coppér
Suiphate is placed at the bottom of the container under ilier paper. This wil
prolong the life of aicohol and itv willturn blue if water is present, wuch is.
indication that fresh alcohol should be used.

4
 3 The dehydatng agents should be chunged whenever they become dilute.
must be agitated in the
4 The tissue
Ahe process.
dehydrating agent to speed up
64 Clearing
The process of clearing involves the rernoval of alcohol and
agents which are not miscible with parafin and rendering the tissueoher dehydratu1g
transparent.
Clearing agents are miscible with both alcohol and paraffin and have high
refractive index whnch make thetissue transparent.
Clearing agents Advantages - Disadvantages
Ceharwood oil 1. Clears alcohol without LNeeds removal by xylene
Relractiveindex 1.50 shrinkage before wax impregnation
2: Clears celloidin 2.Expensive
3. No evaporation 3.Guting is difficult if oil
4. Does nof harden the tissue is not removed
5. Best clearing agent properly
Berzene 1. Clears quickly LHardens the tissue
Refractiveindex 1.50 2. Less shrinkage than 2.Can cause cancer in the
es of xylene laboratory worker
timc' Xylene 1. Clears quickly 1.Hardens the tissue
Refractive index 1.50 2 Makes the tissue 2.More expensive
transparent so that end 3.Not miscible with
point of clearing can be water
made ouf easily 4.Does into dissolve
celloidin
ehlorofom i 1..Tissue can be left longer 1.Toxic and
expensIve
than with other agents
Refractive index 1.45 2. Never over hardens 2.Does not change he
refractive index of tissue
and so end point of
clearing cannot be
made oL.
Aniline oil 1. Tissue can be 1.Toxic
lefl longer than
other agents
2 Cheap, casily available

5
 (vi)
Technique of Clearing
absolute alcohol, thetissue.
of
Afler removing the tissue from the last bath and thcntransferredtO wax,
is placed ìn two changessofxylene for 1/2 to one. hour

L55 Impregnation
Ampregnation with wax
which also
After clearingthetissue isinfilterated with asupporting medium
tissue firm,
eplaces the clearing agent. The supporting medium will make the
facilitates easy sectioning and keeps the various components of the issue n
proper relation. The supporting media used are paraffinwax, paaplast, celloidin
and water soluble waxes. The same agents are used for embedding the tiSsuc.

Epbedding media and their properties

(1) Paraffin wax : Paraffin is solid at room temperature. The mclting point of
paraffin ranges from 45° to 60°C.For tropical countries hard wax having a
melting point of 58-60° is suitable. This is the most commonly used
embedding medium.

(i) Paraplast:Paraplast is amixture of purifiedparaffin and plastic polymers. It

is more elastic and serial sections of 4 micron thickness can be cut easily.

iii) Tissue mat: It is aproduct of parafin and rubber and is similar to paraplast. a3

Gelatin : It is water soluble and so

sary It is used for friable specimensdehydration
and clearing is not neces-
and for frOZen sections, It bas low
mclting point.
/v) Ester wax : Ih is harder than parafiin but has a low
melting
il issoluble in alcohol, so no clearing agent is required point 469-48°C.
of

6
 a melting
(vi) Water soluble :Thesc arc polvelhy icnc glycois having dehydra-
waxes shritnkagethanparafn, and no
pointof 38-42°C. They cayse less
do not :ike the tissues brittle and give
Landclearing is needed. They
useful for denorstration of lipid and en-
better Support than parafin. They are dibCul.
out of sectiornSIS
ynes. They are not used routincly bccsc floating

hard or iragile
(VICelloidin : This is. purified nitroccllulosc, and is uscd forweeksto iny1eg
Speimens. Large specimens can be scctioicd. Ii takes 2-3
slorcd in 70%alcohol.
nate the tissue, with celloidin Blocks should be

Technique of impregnation
be olhiyh quality and should be
used for iupregnation should
raraftin wax meltcd and liltcrzd belos ISC.
free fromn dust, dirtandwater. It is

9
impregnation is kept in the cmtcddinp oven whose temperatue
Paraffin for
point of the wax.
is maintained 2°C above the melting
tissu, Afiei
amount of wax should be 25-30 timcs thc volunç of he
The
placed in the first paraffinwux bath for 2 hours and then in
clearingthetissue is cnsures complcteimpreguatiun.
the second bath for 2hours. This

tissuewhich arc TIOtcasily imprcgnated by routine methods.

Certain dense aprocess callcd vacuum
negative atniospheic prcsuc bv
£re impregnated under
cmbedding.

automatic tissue
of the tissuc can be. donc biliually oi by an
Frocessing
processor.(Fig. I-] and I1-2)
Fig. fek

7
 F

Fig. II-1.Autotechnician

Part of autotechnican (Fig. l-l Autotechnician)

ABody
B-Beaker plat•orm
CThermostatically controlled wax bath
D Beakers
E-Transfer arm
F Metal disc
G--Plastic cover plate
Functions ofAutotechnician
1 The body of an autotechnician moves on rollers which turn thc platform
containing the beakers.
8
 The transfer arm moves the tissue through
it in and out of the bieakers, -sy processing reagents by lifing
Thetiming unit consists of an electric clock to which is
attached amo
disc in which slots are cut at required intervals.
The disc rotates against a spring loaded lever, which, when it
the slot,causes the transfter arm to lft and the slips into
turning
in motion, shifting the tissue to the next position in themechanisn
cycle.
is set

5- Delay mechanism is available to process the tissue at week ends.

Penin.
Parnfin Peralfie

dckaring agem
Ckaring agend
Abmolute akohol

Ahaolule acobol
Aholute alcohot
aknhol

Fg. l-2. Metal disc with slots for 24 hours processing

9
 3
Vll-5)
Metal disc with slots for 24-hours process (Fig.Processing schedule for
4
12 hour processing schedule for manual processing
automatic tissue processor ½ hours
Acetone I
70%alcohol 1 hr ...½ hours 5.
Acetone II
80% alcohol ...1 hr ...4 hous
Acetone III
90% alcohol . 1hr . .½ hours
... 1 hr Acetone Xylol
95% alcohol ...4 hours
Absolute alcohol I ...1 hr XylolI ..½ hours
Absolute alcohol II ...1 hr Xylol I 7.
Paraffin wax I ...2 hrs
Absolute alcohol II . 1hr ...2 hrs
Paraffin wax II
XylolI ..1hr
Xylol II ...lhr
Paraffin waxI ...2 hrs
Paraffin wax II ..2 hrs

Embedding or blocking
tissue in a preciscly
Embedding isthe process of placing the imp1egnatedmedium
embedding and causing
arranged position into a mould containing the
this medium to solidify.
Types of moulds
laid on
"1. Leuckhart's Lpieces:These are Lshaped brass pieces which are
or oblong into which the melted
metal or glass plate to form a squareembedded.
paraffin is poured and the tissue is
also be uscd.
Glass petridishes, metal petridishes and watchglasses can
mould. The tissu
3. Atissue tek consists of plastic rings and a stainless steel
on top, wax isplaced on the.
is placed on the mould, the plastic ring is laiddetached.
is The block coveredl
top of thc ring, cooled and the base mould storage.
and
by the plastic ring is ready for cutting
Technique of Blocking
metal piece.
1. The mould is placed on a tray or
molten wax is pourcd into the mould.
2. Fresh

10
 prèssed to the
forceps and
3 the final wax bath with
Tissue is lifted from downwards.
bottom, with the cutting surlacefacing of the
spccimen is fixcdtothe corner
4. The label bearing the number of the
solidifying wax. thesurface, it is
form a skin on
5. When the block has cooled sufficicntly lo prevent crystallization of
immersed in cold water andcoolcd rapidly lo
Wax.

arerenovcd.
). Whenthë block becomes hard; the noulds
fixcd to the side of the block by press-
7. Afresh label with attyped mumber is
ing a hot scalpel.
 I

SFixation of Tissues
Fixatives
thebody, undergo
The cells and tissues shortly after death or removal from
Autoly-
complete disintegration by a process called autolysis and putrefaction. soon
sis is death of the cells due to the action of enzymes whích. are liberated as of
destruction
as the cell dies, Putrefaction is caused by bacterial invasion and tissue
thetissue. This disintegration of the cellIis prevented by either freezing the
Or addingto it certain chemical substançes, which are called fixatives.
Definition
I
Process by which constituents of cells and tissues are fixed in a physICal
treai
and partly also in a chemical state so that they will withstand subsequent or de
distortion
ment with various reagents with a minimum of loss, significant
composition of tissue.
Aim and functions offixative
rapid
1. Fixative should pevent autolysis and putrefaction ofthe tissue by
coagulation of the cytoplasm.
It should not shrink or swell the cell nor destroy the structure of the
2.
tissue.
the tissue.
3. It should rapidly and evenly penetrate
differentiation.
4. It should give good optical
harden the tissue and render it amenable for easy manipulation.
5. It must
enable ay staining technique to be employed at later date.
6. Itmust
should be cheap, easily available and with long shelf life.
7. It
The classification of fixatives V
and compound fixatives. Simple fixatiyes
Fixatives are classified into: simple chemical action.
the
are further classified as follows, basedon

12
 Aldehydes
1. Formaldehyde..
2-Glutaraldchyde
Oridizing agents
1 Osnium tetraoxide
2 Potassium permanganate
3. Potassium dichromate
1 Protein denaturing age
1 Acetic acid
2 Methyl alcohol
3 Ethyl alcohol
V Miscellaneous
1. Mercuric chloride
2. Picricacid
Simple fxatives their actions and properties
FORMALDEHYDE (H CHO)
Formaldehyde is a gas and when it dissolves in water 40% weight by
volume(whv) it is called formalin. Formaldehyde is in polymerisedform in fornali.
and is not suitable for fixation of the tissse. It is diluted to 10% formalin when
depolymerisation occurs and it becomes suitable for fixation.
Mode of action
1. It forms cross links between amino acids of protein and renders them in
soluble.
2 Italso fixes lipids for frozen section.
3. Itpenetrates rapidly and never Overhardens the tissue.
4. It is cheap and permits the use of various staining procedures.
5 Itis ideal for mailing the specimens.
Fixation time-4-5 mm thick tissue is fixed in 8hours.
Disadvantages
1
lU irmitates the nose, eyes and skin. Hence adequate ventilation and the use
of gloves are essential.
22, Itforms awhite precipitate of paraformaldehyde which can be prevented by
adding methanol.
13
 neutralizedby.adding 2% cal-
can be
3. Formalin contains formic
acid whichformation ofblack< formalin
pigment in
preventsthe
cium acetate which also
the sections.
GLUTARALDEHYDE(CH CHOCHO) fxes by
microscopy. It also cross linking
This is usually used for electron the tissue.
with protein and collagen. It rapidly: fixes
Disadvantages
1 Itis expensive.
2. Its penetration is poor.
3. It overhardens the tissue.
toSMIUMTETRAOXIDE (OO)
l. Itisused for Fixationfor electron microscopy.
It demonstrates lipids by being reduced by them to from a black com
2.
pound and forms cross links with protein.
the tissue soft and friable.
3. Itis expensive, penetrates poorly and leaves
4. It irritates the eyes.
POTASSIUM DICHROMATE
This fixes cytoplasn without precipitaion of protein.
L Itforms chromium ions which along with water, combine with carboxy1
and hydroxyl groups of protein and fix the cytoplasm.
2. It is unsuitable for histochemistry for theabove reason.
3. Itis ideal for fixingmitochondria and lipids.
4. After fixation, the tissue should be thoroughly washed in running waler
overnight to prevent the formation of insoluble precipitate. to
th
ACETICACD
L Itisaprotein prcipitant.
2 It swells the tissue and hence is used only in compound ixauve.
ETHYLAOCOHOLAND METHYL ALCOHOL
1. Itdenatures proteins and precip1tates them.
14
 leaves them in
2. It is useful for histochemical mcthod for chzymcs since it
the original state,,
3. It is a fat solvent.
hence, is used in a
4. It penetrates slowly and hardcns the lissuc and
compound fixative.
5. Itis also employed as a cytological fixative.
MERCURICCHLORIDE
1. It precipitates protein.
2. It penetrates thetissue rapidly and hardens il.
3. It shrinks the tissue.
following mercuric chlorik
4. Cytoplasmic staining becomes morc brilliant
fixation.
5. Itforms a browish mercury prccipitate in thç Ússuc. This is removed b,
followed by sod1ur
treating the section in iodine, then in alcoholcombines with sodiur
hiosulphate. Iodine forms mercuric iodide which However, it is toxit
thiosulphate to form soluble mercuric tctrathionate. chloride.
le. by zinc
and corrodes gold and, hence, it is rcplaccd
PÍCRICACID
1 Itprecipitates protein.
for trichrome stain.
2. Itgives a brilliant contrast removes iron from thetissue.
3. It lakes red blood cells and
yl fixativc.
4. Itis suitable asa compound should be slored under layer ofwat
it
5. Itis explosive in dry state, hencc
Ncompound fixatives
the product of two or inore simplc fixatives mi.
ter Compound fixatives are are classified in
effect of tlhcir propcrlics. They
together toobtain the combined cytological arnd histouhemicai fixai:
microanatomical,
three groups, namely,
Microanatomical fixatives
large specimcns where the cells and ix
1 They are used to preserve
relationship is to be maintaincd.
2 Jhey are used routincly.
15
 3. They nevcr ovcrharden the tissue FOR
10%FORMALIN
Fonr
Formula
Fornula (40% Formaldehyde in water)
...10ml.
Water
...90 Im
Advantages 1.
1. It is ideal for surgical and postmortem specimens. 2.
2. It causes even fixation and produces very litde shrinkage. -HIE
3. Tissue can be kept infinitely in it. Fo
4. It forms the basis for museum fixatives.
Disadvantages
1. It is a slow fixative.
2. It forms black formalin pigment with blood.
3. It is an irritant.
Fixation tome: 5 mm thick tissue is fixed in 6-12 hours at room temperature.
10%FORMAL SALINE
tis
Formula 1.
Formaldehyde 40% b. 100ml
2
Sodium chloride ...9 gm. 3.
Distilled water ...900 ml.
4
1 It is ideal for fixing the brain.
5.
2. The period of fixation of large tissue is 24 hours.
10%BUFFERED FORMALIN
F
Formula
...10 ml.
Formaldehyde 40% (Formalin)
Sodium dihydrogen phosphate ..0.4 gm.
..0.65 gm.
Disodium hydrogen phosphate (anhydrous) ..90 ml.
Distilled water
1. Itprevents the formation of formalinpigment.

16
FORMOLCALCIUM
Fomula
Formalin ... 10ml.
Calcium acetate ...2gn.
Water up to 100 ml.
1.1. It preserves phospholipids.
2. It prevents the formation of formalin pigment
-HIEDENHAN'S SUSA
Formula
Mercuric chloride
Sodium chloride
45 gm.
5gm.
Trichloracetic acid ..
20 gm.
Glacial acetic acid 40 ml.
Formaldehyde 40% 200 ml.
Distilled water 800 mi.
Fixation time: 12-24 hours for 7-8 mm thick tissue, 2-3 hours for 3mmthick
tissue.
1. Itallows brilliant staining.
2 Itpenetrates rapidly and fixes evenly.
3. Itis intolerant fixative ifkept for more than 24 hours.
4 Tissue must be directly transferred to 96% alcohol.
5. Tissue should be treated with iodine to remove mercury pigment.
ZENKER'SFLUID
Formula
Mercuric chloride ... 5gm.
Potassium dichromate ... 2.5 gm.
Sodium sulphate ... gm.
Distilled water upto 100ml.
Add glacial acetic acid immediately
before use 5ml.
Fixation time 12-24 hours. 3mm tissue fixes within 2-3 hours.
2.
It enables excellent stainin:: of connective tissue fibres.
17
 3. Its penctration is poor. washedin running water overnight to re-
4. Afler fixation, tissuc should bc pigmentshouldalsobe removed by treat-
move excess dichromate. Mercury
ing with iodine.
ZENKERFORMOL (HELLY'S FLUD)
Formula . 5gm.
Mercuric chloride ...
2.5 gm
Potassium dichromate ..
lgm.
Sodium sulphate 5 ml.
Distilled water
Add formalin before use
1. Fixation time:6-24 hours.
containing organs.
2 It is an excellent fixatiye for nucleus and blood
3 Itis slow in fixation.
as for
4 Mercury pigment and potassium dichromate should be removed
Zenker's fluid.
Bouin's fluid
Formula
Saturated aqueous picric acid 75 ml.
Formaldehyde (40%) 25 ml.
Glacial acetic acid 5ml.
1. Fixation in 24 hours; 2-3 mm thick tissue in 2-3 hours.
2 It penetrates rapidly and evenly.
3. It gives brilliant staining by trichrome stain.
4. It preserves glycogen.
5 Tissue should be transferred directly to 70% alcohol,
GENDRESFLUD
Formula
Picric acid satrurated in 95% alcohol ..80 ml
Fornalin (40% Formaldehyde) .15 ml
Glacial acetic acid .5ml
Tts properties are same as the Bouin's fluid. It is the ideal fixative for
glycogen.
18
 Slnudve

Cytological Fixatives
Cytological fixatives atc divided into nuclcat and cytoplasmic fixatives
ANuclear Fixatives
CARNOYSFLUID
Fomula
Absolute alcohol ...60 ml
...30 ml.
Chloroform
...10 ml
Glacial acetic acid
1 Fixation time: ½to 3hours.
2. Itis ideal for fixingchromosomes, lynph nodcs, urgCnt biopsiesnd glycog
3. Itis a poor cytoplasmic fixative.
4. After fixation, tissue is transferred dircctly to absolute alcohol.
CLARKESFLUD
Formula
...75 ml.
or
Absolute alcohol
...25 ml.
Glacial adetic acid
1. Fixationtime:1-2 hours.
2. It penetrates rapidly.
3. It is agood nuclear as well as cytoplasmic fixative.
E4. Itis ideal for Smears.
Cytoplasmic Fixatives.
CHAMPYS FLUD
Formula
3% Potassium dichromate ...7ml.
1% Chromic acid ...7 mi.
2%Osnuim tetroxide ... 4 ml/
E1. Fixationtime:12 hours for 2mm thick tissuc.
2 It preserves mitochomdria, and lipids,
3. It penetrates poorly.

19
 Formal saline, and formal calcium and Zenker's formol
cytoplas1mic fixatives. are go0

Mistochenical fixatives is
str
These fixatives are used when it becomes essential to demostrate
enzyme
and other chemical substances of the cell. These fixatives should not alter th
normalchemistry and should not interfere with the staining procedures.
1 With buffered formalin as a fixative many histochemical techniques can b nE
performed.
2. Imnersion in cold acetone at 0-4°C is used to demonstrate phosphatases.
St
3 Absolute alcohol can be used as an alternative to buffered formalin.

The other methods of fixation are vapour fixation, secondary fixation

postchromatization and freeze drying. Formadehyde. Acetaldehyde and Glutaral
dehyde vapours are employed to fix cryostat cut sections.
Secondary fixation
Zenker a 2
This involves fixing the tissue first in formalin followed by
Heidenhain's susa.

It gives a texture to the tissue andimmersedimproves staining qualities. I 3

postchromatization, formalin fixed tissue is in potassium dichromat
for several hours to mordant the tissue and enable good mitochondria
solution
staining.
the tissue is frozen drV sothat there is no alteration in th
Infreeze drying,
structure or chemistry, followed bywax embedding.

20