CIHRT Exhibit P-3359 Page 1 Histopathological techniques Histopathological techniques The adoption of routine fixation and paraffin wax The adoption of routine fixation and paraffin wax embedding. embedding. ‘Routine’ tissue preparation in modern ‘Routine’ tissue preparation in modern diagnostic histopathology. diagnostic histopathology. 1743 Baker - Alcohol preservation of Hydra 1743 Baker - Alcohol preservation of Hydra 1851 Clarke - Alcohol/acetic as fixative 1851 Clarke - Alcohol/acetic as fixative 1879 Fredericq - Alcohol/turpentine/wax (evaporated) 1879 Fredericq - Alcohol/turpentine/wax (evaporated) 1881 Giesbrecht - Alcohol/turpentine/molten wax 1881 Giesbrecht - Alcohol/turpentine/molten wax 1893 Blum - Formaldehyde as fixative 1893 Blum - Formaldehyde as fixative 1899 Hardy - Studied variety of fixative/wax processes 1899 Hardy - Studied variety of fixative/wax processes Bryan R. Hewlett ART, MLT. Bryan R. Hewlett ART, MLT. 1905 Brasil - Formaldehyde/alcohol/acetic fixative 1905 Brasil - Formaldehyde/alcohol/acetic fixative 1880 - 1910 - Medical schools include microtechnique 1880 - 1910 - Medical schools include microtechnique 1910 - 1950 - FFPE becomes prevalent technique 1910 - 1950 - FFPE becomes prevalent technique * Bracegirdle 1987 * Bracegirdle 1987 1 2 Fixation Fixation The influence of processing The influence of processing Fixation is the single MOST important Fixation is the single MOST important preparative histological technique. preparative histological technique. Dependent on quality of fixation Dependent on quality of fixation Poor fixation CANNOT be remedied at Poor fixation CANNOT be remedied at any later stage. any later stage. 3 4 1
CIHRT Exhibit P-3359 Page 2 Dehydration effects Dehydration effects Processing of tissues for Processing of tissues for histological analysis Following optimal fixation in NBF, Following optimal fixation in NBF, histological analysis ethanol removes some lipids and a few proteins ethanol removes some lipids and a few proteins not immobilized by cross-linking. This can not immobilized by cross-linking. This can produce a small amount of tissue dependent produce a small amount of tissue dependent Dehydration Dehydration shrinkage (2 -15%). Some hardening also occurs. shrinkage (2 -15%). Some hardening also occurs. Intermediate solvent (Clearing) Intermediate solvent (Clearing) Infiltration with support media Infiltration with support media Ethanol fixation produces tissue dependent Ethanol fixation produces tissue dependent shrinkage of 35 - 40% and much more shrinkage of 35 - 40% and much more hardening!!!! hardening!!!! 5 6 Paraffin wax effects Paraffin wax effects Intermediate solvent effects Intermediate solvent effects Removes lipids and causes some hardening. Removes lipids and causes some hardening. The heated wax causes the majority of tissue The heated wax causes the majority of tissue Xylene is a ‘true’ clearing agent i.e. it Xylene is a ‘true’ clearing agent i.e. it shrinkage (may total 30-40%). shrinkage (may total 30-40%). raises the R.I. of tissue. raises the R.I. of tissue. May be reduced by minimizing the heat shock May be reduced by minimizing the heat shock It also removes some lipids, causes some It also removes some lipids, causes some on transfer from xylene to molten wax. on transfer from xylene to molten wax. shrinkage and also some hardening. shrinkage and also some hardening. (Time may be shortened by agitation and (Time may be shortened by agitation and negative pressure) negative pressure) 7 8 2
CIHRT Exhibit P-3359 Page 3 Effects of fixation/processing Effects of fixation/processing Loss of constituents Loss of constituents Shrinkage Shrinkage Hardening Hardening Change in optical properties Change in optical properties Inactivation of most enzymes Inactivation of most enzymes Change in acidophilic/basophilic properties Change in acidophilic/basophilic properties Destruction or masking of antigen epitopes Destruction or masking of antigen epitopes Change in morphology Change in morphology ALL OF THESE EFFECTS ARE ALL OF THESE EFFECTS ARE MINIMIZED FOLLOWING OPTIMAL MINIMIZED FOLLOWING OPTIMAL FORMALDEHYDE FIXATION!! FORMALDEHYDE FIXATION!! 9 The nature of fixatives The nature of fixatives Non-Coagulant fixatives Coagulant Fixatives Non-Coagulant fixatives Coagulant Fixatives Formaldehyde Alcohol Formaldehyde Alcohol Protein secondary structure Protein primary structure Protein secondary structure Protein primary structure intact. Only modifies tertiary intact. Only modifies tertiary intact. Alters secondary and intact. Alters secondary and and quaternary structures, tertiary structures, and quaternary structures, tertiary structures, (Methylene bridge cross-links) (Methylene bridge cross-links) (Hydrophilic/phobic inversion) (Hydrophilic/phobic inversion) mostly (90%) retrievable, often irretrievably, with loss mostly (90%) retrievable, often irretrievably, with loss with little loss (<1%) with little loss (<1%) of up to 40% of protein. of up to 40% of protein. of protein. of protein. 12 3
CIHRT Exhibit P-3359 Page 4 Formaldehyde fixation Formaldehyde fixation Non-coagulant fixative Non-coagulant fixative Fast penetration Fast penetration Slow fixation Slow fixation Little loss of constituents (< 1%) Little loss of constituents (< 1%) Little shrinkage Little shrinkage ‘Soft’ fixative ‘Soft’ fixative Many effects are reversible Many effects are reversible Most realistic overall morphology, Most realistic overall morphology, allows widest range of histochemistry allows widest range of histochemistry Ileum – NBF fixed section Ileum – Alcohol fixed section Ileum – NBF fixed section Ileum – Alcohol fixed section 13 14 Fixation Fixation Alcohol fixation Alcohol fixation Reality #1 Reality #1 Coagulant fixative Coagulant fixative Medium penetration (K = 1.0) Medium penetration (K = 1.0) Formaldehyde fixation provides the most Formaldehyde fixation provides the most Fast fixation (fixes as it penetrates) Fast fixation (fixes as it penetrates) Loss of constituents ( ↑ 40%) Loss of constituents ( ↑ 40%) realistic overall morphology and becomes realistic overall morphology and becomes Causes shrinkage Causes shrinkage the standard fixative for the majority of the standard fixative for the majority of ‘Hard’ fixative ‘Hard’ fixative routine diagnostic histopathologists! routine diagnostic histopathologists! Not readily reversible Not readily reversible Great for nuclear morphology and staining of Great for nuclear morphology and staining of nucleoproteins, restricts range of histochemistry nucleoproteins, restricts range of histochemistry 15 16 4
CIHRT Exhibit P-3359 Page 5 ‘Routine’ tissue preparation ‘Routine’ tissue preparation Development of tissue preparative techniques Development of tissue preparative techniques The current state of the art The current state of the art Fixation re-visited Fixation re-visited Effects on staining Effects on staining Effects on QA Effects on QA What we need to do to improve What we need to do to improve Formaldehyde allows the widest range of histochemical stains Formaldehyde allows the widest range of histochemical stains 17 18 Formaldehyde fixation – automated processing Formaldehyde fixation – automated processing 1950’s processor (optional 24 hour and 7 day timers) 1950’s processor (optional 24 hour and 7 day timers) 19 20 5
CIHRT Exhibit P-3359 Page 6 Fixation Fixation Reality #2 Reality #2 Following formaldehyde fixation; Following formaldehyde fixation; automated processing techniques provide automated processing techniques provide an advantage in speed and ease of use, an advantage in speed and ease of use, with no loss of morphology! with no loss of morphology! Modern closed processor (flexible timer and pressure/heat control) 21 Modern closed processor (flexible timer and pressure/heat control) 22 Fixation Fixation Reality #3 Reality #3 Formaldehyde fixation becomes Formaldehyde fixation becomes integrated with automated processing, integrated with automated processing, providing a further advantage in speed providing a further advantage in speed and ease of use. and ease of use. Formaldehyde fixed Formaldehyde fixed Today’s “Routine” Today’s “Routine” automated processing automated processing The change in morphology is deemed The change in morphology is deemed acceptable and becomes ‘Routine’! acceptable and becomes ‘Routine’! 23 24 6
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