HEMATOXYLIN STAIN.

ITS
IMPORTANCE IN HISTOLOGY
Dr.Pankaj Kaul
Deptt. Histopathology
PGIMER, CHANDIGARH
INTRODUCTION
Hematoxylin and Eosin (H&E) stain is probably
the most widely used stain due to its;
· Comparative simplicity
· Ability to demo. clearly the diII. Tissue
structures
H is extracted Irom the heart wood 'LOG WOOD¨
oI the tree Haematoxylin campechianum that
originated in Maxican state oI Campeche, but is
now mainly cultivated in the West Indies.
EXTRACTION OF HEMATOXYLIN
· H is extracted Irom Log wood with hot water and
then precipitated out using Urea.
· H itselI is not a stain. The maior oxidation product
in it is HEMATIN, a natural dye that is responsible
Ior the color.
· Hematin is produced Irom H in two ways;
Natural oxidation (ripening) by exposure to
light and air. It is slow process and takes 2-3 mo. e.g
Ehrlich`s and DelaIield`s H (long shelI liIe)
Chemical oxidation using Sod.Iodate e.g Mayers`
& Harris`s H. Cont..
CHEMICAL OXIDATION
· Chemical oxidation converts H to Hematin instantly and
is ready to use immediately aIter preparation .
· These have a shorter shelI liIe than naturally oxidised H.
· Hematin is anionic (vely charged) & has poor aIIinity
Ior tissue. It thereIore needs the presence oI a mordant.
· The most useIul mordant Ior H are salts oI Aluminium,
Iron, Tungsten, Molybdenum & Lead.
· The mordant/ metal cation conIers a net ¹ve charge to the
dye-mordant complex and enables it to bind to anionic
tissue sites.
· Most mordants are incorporated in, into H staining sol.,
while some need pretreatment with mordant beIore staining
(Heidenhain`s Iron H) Cont..
CLASSIFICATON OF HEMATOXYLIN
· H can be classiIied according to mordant used;
Alum Hematoxylin
Iron Hematoxylin
Tungsten Hematoxylin
Molybdenum Hematoxylin
Lead Hematoxylin
Hematoxylin without mordant
ALUM HEMATOXYLIN
· Is the one used routinely in H&E staining
· Mordant is Aluminium in the Iorm oI Potash
alum or ammonium alum.
· All stain nuclei red which is converted to blue-
black on washing with alkaline sol. (Tap water)
· Scott`s tap water is also used Ior this purpose
called as 'Bluing¨
· Alum H can be used Regressively i.e over
staining and then diIIerentiating in Acid alcohol
Iollowed by bluing or Progressively i.e stained Ior
a predetermined time Ior optimum staining. Cont..
ALUM HEMATOXYLIN
· The time may vary depending on type oI tissue or
age oI H
· The most commonly used H in H&E are;
Ehrlich`s H
Mayer`s H
Harris`s H
Gill`s H and
DelaIield`s H
Carazzi`s H is used particularly Ior urgent Frozen S
EHRLICH`S HEMATOXYLIN 1886
· It is a naturally ripening pot.alum H, which
takes 2 mo to ripen.
· Ripening is shorter in summer sun and prolongs
in winter cold.
· Once ripened the stain works Ior many years.
· Being an excellent nuclear stain, it also stains
mucin including mucopolysaccharides oI cartilage
· Recommended Ior Bone, Cartilage and Acid
Iixative Iixed tissues(Bouin`s)
· Once stained does not Iade & stays Ior long.
PREPARATION OF EHRLICH`S STAIN
Hematoxylin 2gm
Ab. Alcohol 100ml
Glycerin 100ml
D. Water 100ml
G.Acetic acid 10ml
Pot. Alum 15gm
· H is dissolved in alcohol and other chemicals are
added. Glycerin is added to slow down oxidation
process and prolong shelI liIe.
· To shorten ripening time 50mg oI Sod.iodate is
added. Always Iilter beIore use.
DELAFIELD`S HEMATOXYLIN 1885
· It is naturally ripened Am alum H with similar
longevity as Ehrlich`s H stain.
· Here saturated aqueous Am. Alum is used.
MAYER`S HEMATOXYLIN 1903
· It is an Pot/Amm alum H, chemically ripened with
Sod. Iodate
· Can be used as regressive as well as progressive
stain
· It is used to demo Glycogen in various Enzyme
histochemical methods
· The stain is applied Ior short time(5-10min) till
nuclei are stained and then 'Blued¨ without any
diIIerentiation.
· An additional charcol hydrate(5gm) and Citric
acid (1gm) are added and boiled Ior 5min cooled &
Iiltered.
HARRIS`S HEMATOXYLIN 1900
· A Pot. Alum H, traditionally ripened with mercuric
oxide(Toxic/ environ.unIriendly with detrimental/
corrosive eIIect on automated staining machine)but
now Sod.or Pot. Iodate is substituted Ior oxidation.
· Harris`s is a useIul general purpose H giving clear
nuclear staining used as progressive stain in exIoliate
cytology & regressively Ior histology.
· As with most chemically ripened alum H, the
quality oI nuclear staining begins to deteriorate in Iew
months. Staining time may be increased accordingly.
· Fresh batch may be prepared monthly.
COLE`S HEMATOXYLIN 1943
· A Pot alum H, is artiIicially ripened with an
alcoholic iodine sol.
CARAZZI`S HEMATOXYLIN 1911
· Is an Pot alum H, which is chemically ripened
using Pot iodate. Like Mayer`s H, Carazzi`s H may
be used Ior a progressive nuclear counter stain
using short time, Iollowed by bluing in T.W.
· It does not stain any cytoplasmic components.
Due to short staining time it is useIul Ior Frozen
sections.
GILL`S HEMATOXYLIN
· Double or triple H concentration may be used as
preIerred and are reIerred to as Gill`s1(normal),
Gill`s2 (double) & Gill`s3 (triple) H.
· Gill`s H is more Irequently used Ior routine H&E
than Mayer`s H and is more stable than Harris`s
H, as oxidation is inhibited to the extent that no
measurable change occurs over many months.
· The only disadv. OI this H is that it stains gelatin
adhesives, Glass slide and some mucus also
STAINING TIMES WITH ALUM
HEMATOXYLIN
STAINING TIME IN MINUTES
Cole`s 20-45
DelaIield`s 15-20
Ehrlich`s (Progressive) 20-45
Mayer`s (Prog.) 10-20
Mayer`s (Regressive) 5-10
Harris`s (Prog.) 4-30 Seconds
Harris`s (Reg.) 5-15
Carazzi`s (Prog.) 1-2
Carazzi`s (Reg.) 45 seconds
Carazzi`s (Frozen section) 1
Gill`s (Reg.) 5-15
THUMB RULE FOR HEMATOXYLIN
STAINING
As a general rule the time need to be;
· Shortened Ior Frozen sections and
· Increased Ior decalciIied tissues and those that
have been stored Ior a long time.
DISADVANTAGES OF ALUM
HEMATOXYLIN STAIN
· The maior disadvantage oI alum H nuclear stain is their
sensitivity to any subsequently applied acidic stain e.g
van Gieson and other Trichrome stains.
· The application oI Picric acid-acid Fuchsin mixture in van
Geison`s stain removes most oI H and renders nuclei
invisible. In such case Iron-mordant H e.g Weigert`s H
which is resistant to the eIIects oI Picric acid is used.
· A new popular alternate is the, combination oI Celestine
blue stain sol with an alum H to undo the eIIect oI acidic
stains and strengthen the bond between nucleus and alum
H and to give stronger stain resistant to acids.
EOSIN STAIN
· Is the most suitable stain to combine with
an alum H to demonstrate general
histological architecture oI a tissue.
· Its` value is due to its` ability to distinguish
between cytoplasm oI diII. Cells and
connective tissue Iibers by staining them
with diII. Shades oI red and pink.
PROPERTIES, TYPES OF EOSIN
Eosins are Xanthene dyes, commercially available as
· Eosin Y (water soluble)
· Ethyl Eosin (alcohol soluble) and
· Eosin B
oI these Eosin Y is most widely used eosin.
· As cytoplasmic stain it is used as 0.5° or 1° sol.
In D.W with a crystal oI Thymol to inhibit Iungal
growth.
· Addition oI little Acetic acid sharpen the staining.
· DiIIerentiation oI Eosin occurs by T.W wash
Iollowed by dehydration with subsequent alcohols.
STAINING METHOD USING ALUM
HEMATOXYLIN
· Dewax and BSW
· Remove Iixation pigments (by saturated alcoholic
Picric acid sol.which may have deleterious eIIect on
subsequent staining)
· Treatment with 10° Am. Hydox. In 70° alcohol
Ior 5-15 min. which is comparatively a saIe method.
· Stain with alum H oI choice and wash with T.W
'Bluing¨ Ior 5 min.
· DiIIerentiate in 1° acid alcohol (1°HCl in 70°Al.)
Cont..
STAINING METHOD USING ALUM
HEMATOXYLIN
· Wash in T.W Ior 10-15 min.
· Dip in Alk.Ammonia water/ Scott`s T.W Ior 5 min.
· Stain in 1° Eosin Ior 10 min and wash in T.W Ior 1-5
min.
· D.C.M in DPX
Results:
Nuclei Blue/ Black
Cytoplasm Varying shades oI pink
Muscle Iiber Deep pink/red
RBC Orange/ Red
Fibrin Deep pink
IRON HEMATOXYLIN
· In these Hs, Iron salts are used both as oxidising
agent and as mordant.
· The most commonly used Iron salts are Ferric
chloride and Ferric Amm. Sulphate.
· Commonly used I.H are;
· Weigert`s H
· Heidenhain`s H
· Loyez H Ior Myelin
· VerhoeII`s H Ior elastin Iibers
cont..
IRON HEMATOXYLIN
· Over oxidation oI H is a problem with these
stains. So it is usual to prepare separate mordant/
oxidant and H sol and mix them iust beIore use
(Weigert`s H) or to use them consecutively
(Heidenhain`s H)
· IH are capable oI demonstrating a much wider
range oI tissue structure than AH, but the
technique is more time consuming and needs M/E
to evaluate accurate diIIerentiation.
WEIGERT`S HEMATOXYLIN 1904
It is an IH with Ferric chloride as
mordant/oxidant.the iron and H are prepared
separately and mixed beIore use;
A. Hematoxylin sol:
Hematoxylin 1gm
Ab.alcohol 100ml
Allowed to ripen naturally Ior 4 wks beIore use.
B. Iron sol:
HCl (Conc) 1ml
D.W 95ml
WEIGERT`S HEMATOXYLIN 1904
· Sol is Iiltered and mixed equal vol oI I&H
beIore use. The mixture should be violet black,
but iI it turns brown should be discarded.
· The main use oI WH is as nuclear stain where
acidic solutions are to be applied subsequently
e.g van Gieson`s stain
HEIDENHAIN`S HEMATOXYLIN 1896
· Is IH using Ferric amm. Sulphate as
oxidant/mordant and the same solutions is used as
diIIerentiating Iluid.Iron sol. is used Iirst and then
H sol. till over stained Iollowed by diIIerentiation
with iron sol under microscope control.
· It stains Mitochondria, Muscle striations, Myelin
& Chromatin etc. Grey black
· Section stained with H`s IH stain are resistant to
Iading, only iI section is washed well aIter
diIIerentiation to remove all traces oI iron alum.
LOYEZ`S HEMATOXYLIN 1910
· It is a IH with Ferric amm sulphate as mordant
· The mordant is used consecutively and
diIIerentiation is by Weigert`s diIIerentiator.
· It is used to demonstrate Myelin
Its two methods similar to Loyez are;
. Heidenhen`s Myelin stain is similar as Loyez
but Ior iudicious selection oI staining time
omits diIIerentiation step.
2. Short Weil technique in which mordant & dye
are mixed beIore use rather than consecutively.
VERHOEFF`S HEMATOXYLIN 1908
· It is IH, used to demonstrate Elastic Iibers.
· Ferric chloride with Lugol`s Iodine is used
as mordant and 2° Ferric chloride is used
as diIIerentiator.
· Coarse elastic Iibers stain --------- Black
TUNGSTEN HEMATOXYLIN 1969
· There is only one widely used TH though there
are many variants on the original Mallory
PTAH technique.
· Mallory (1897,1900) combined H with 1° aq.
Phosphotungstic acid acting as mordant.
· Hematoxylin can be replaced by hematein.
· It is used in CNS and general tissue structure
demonstration.
Cont..
TUNGSTEN HEMATOXYLIN 1969
· Muscle striations, Dark Blue
Neuroglia Iibers, Fibrin
& Amoeba
· Nuclei,Cilia, RBC Light blue
· Collagen,asteiod, Deep brown-red
cartilage & elastic Iiber
· Cytoplasm Pale pinkish-brown
MOLYBDENUM HEMATOXYLIN 1941
· H sol using Molybdic acid as mordant are rare
and the only technique which gained acceptance
was Thomas (1941) technique.
· The method is recommended Ior demonstration
oI Collagen and coarse reticulin in addition to
ArgentaIIin cell granules.
Collagen & coarse reticulin Violet black
ArgentaIIin cells Black
Nuclei Pale blue
LEAD HEMATOXYLIN
· H sol with lead salts have recently been used in
demonstration oI granules in endocrine cells oI
alimentry tract etc.
· The most practical diagnostic application oI it is in
identiIication oI endocrine cells in tumors oI
doubtIul origin and in research it is used to
localize gastrin secreting cells in stomach by
Beltrami method 1975.
· The most reliable method Ior investigation oI
diIIuse endocrine system is by Solcia method
1969.
HEMATOXYLIN WITHOUT MORDANT
· Freshly prepared H sol without mordant have
been used to demonstrate various minerals in
tissue sections like Lead, Iron and Copper etc.
· Mallory 1938 described a method to demo
Lead and later published similar method to
demo Iron and Copper
· The basis oI Mallory method is the ability to oI
un-ripened H to Iorm blue-black lakes with
these metals.
HEMATOXYLIN STAIN AT A GLANCE
Mordant Oxidant Example Applications
Alum Natural Ehrlich Nuclear stain
Alum Natural DelaIield Stains some mucin
Alum Sod.iodide Mayor Nuclear used with Eosin
Alum
Mercuric Chloride
Harris
Nuclear used with Eosin
Nuclear counter stain
Alum Iodine Cole Nuclear used with Eosin
Alum Pot.iodate Carazzi Nuclear used with Eosin
Alum Sod iodate Gill Nuclear used with Eosin
Iron Natural Weigert Nuclear used with acid dyes
Iron Natural
Heidenhain Intranuclear detail, muscle striations
Iron Natural VerhoeII Elastic Iibers
Iron Natural Loyez Myelin
Tungsten Natural Mallory
PTAH
Fibrin, Muscle striations, Glial Iibers
Molybdenum
Hydrogen peroxide
Thomas
Collagen, endocrine cell granules
Lead Solcia Endocrine cell granules
Without Mordant
Mallory Iron, copper and Lead
NIPS