Frozen Section & Stains Flashcards
Fresh frozen Tissue
Fresh tissue if removed e.g. lymph nodes, suspicious areas, margins, unexpected findings
Sent to …..
Rapid processing (<30min)
Pathologist selects areas/dissects received tissue …………. (biopsy or small representative resections)
Biomedical Scientist (BMS) cuts sections using …… …………….
BMS stains the sections with rapid H&E
Pathologist examines sections and …………..
Fresh tissue is removed e.g. lymph nodes, suspicious areas, margins, unexpected findings
Sent to pathology
Rapid processing (<30min)
Pathologist selects areas/dissects received tissue sample (biopsy or small representative resections)
Biomedical Scientist (BMS) cuts sections using Cryostat microtome
BMS stains the sections with rapid H&E
Pathologist examines sections and reports
fresh frozen applications
Diagnostic relevance
- S……. ……. …..
.e.g. breast cancer, head & neck cancers - T…….. ………
e. g. eye and adnexa regions - basal cell carcinoma
Sentinel Lymph nodes
e.g. breast cancer, head & neck cancers
Tumour margins
e.g. eye and adnexa regions - basal cell carcinoma
Example of Sentinel Lymph Node (SLN) Dissection
Definition
gate-keeper” or the first ….. to drain the tumour e.g. primary breast cancer.
Focuses on identifying ……-……….. patients to avoid unnecessary node dissection.
Focuses on identifying ……-……….. patients to complete node dissection, prescribe systemic therapy.
To avoid Lymphedema (tissue swelling). In the case of extensive lymph node ……. in an armpit, the swelling may affect an ……. …… In addition, there is an increased risk of infection in the affected area.
Ways to check lymph nodes
A……..lymph node d……….
Se……… lymph node b……
gate-keeper” or the first node to drain the tumour e.g. primary breast cancer.
Focuses on identifying node-negative patients to avoid unnecessary node dissection.
Focuses on identifying node-positive patients to complete node dissection, prescribe systemic therapy.
To avoid Lymphedema (tissue swelling). In the case of extensive lymph node surgery in an armpit, the swelling may affect an entire arm. In addition, there is an increased risk of infection in the affected area.
Axillary lymph node dissection
Sentinel lymph node biopsy
Example SLNB Breast cancer
A surgeon injects a ra………. substance, a blue dye, or both near the tumour to locate the position of the ……. lymph node. The surgeon then uses a device that detects ……… to find the ……. node or looks for lymph nodes that are stained with the blue dye. Once the sentinel lymph node is located, the surgeon makes a small incision (about 1/2 inch) in the overlying skin and removes the ……
The sentinel node is then checked for the presence of ……. cells by a pathologist. If cancer is found, the surgeon may remove additional …….. nodes, either during the same biopsy procedure or during a follow-up surgical procedure.
A surgeon injects a radioactive substance, a blue dye, or both near the tumour to locate the position of the sentinel lymph node. The surgeon then uses a device that detects radioactivity to find the sentinel node or looks for lymph nodes that are stained with the blue dye. Once the sentinel lymph node is located, the surgeon makes a small incision (about 1/2 inch) in the overlying skin and removes the node.
The sentinel node is then checked for the presence of cancer cells by a pathologist. If cancer is found, the surgeon may remove additional lymph nodes, either during the same biopsy procedure or during a follow-up surgical procedure.
Example of Sentinel Lymph Node (SLN) Detection
SLN Detection with Fluorescence Imaging for ……. and ……… Cancer
For example, the fluorescent dye indocyanine green (ICG) makes the SLN …..like a firefly under … imaging.
When performing surgeries for endometrial cancers, oncologists must determine whether cancer has ….. to……. …….. to identify the optimal post-adjuvant therapy.
Recent research has shown that over 20 percent of patients who have a full lymph node dissection may develop lifelong ………….
By reducing the number of nodes r…….., SLNB reduces ……. ……., complication rates and morbidity, and significantly reduces risk for lymphedema
SLN Detection with Fluorescence Imaging for Uterine and Cervical Cancer
For example, the fluorescent dye indocyanine green (ICG) makes the SLN glow like a firefly under NIR imaging.
When performing surgeries for endometrial cancers, oncologists must determine whether cancer has spread to lymph nodes to identify the optimal post-adjuvant therapy.
Recent research has shown that over 20 percent of patients who have a full lymph node dissection may develop lifelong lymphedema.
By reducing the number of nodes resected, SLNB reduces surgical times, complication rates and morbidity, and significantly reduces risk for lymphedema.
Fresh Frozen Other Applications
Immuneflurorescence Skin
- Investigation of Immunoglobulin …..
- ……. labelled with fluorescent dye to mark antigens in ……
- Antibodies used:
Ig.., Ig.., Ig…, C3c and fib……. - Formalin fixative ……. used due to tissue autofluorescence under ultraviolet light
- Pemphigoid / Pemphigus
Investigation of immunoglobulin deposition
Antibodies labelled with fluorescent dye to mark antigens in tissue
Antibodies used:
IgG, IgA, IgM, C3c and fibrinogen
Formalin fixative NOT used due to tissue autofluorescence under ultraviolet light
Pemphigoid / Pemphigus
Fresh Frozen Other applications
Immunochemistry
- ….. …….. e,g, cytomegalovirus, herpes virus. advenovirus
- Testing of a …. ……..
- Preserved ………. activity
- Phosp………. detection
Viral infections e.g. cytomegalovirus, herpes virus, adenovirus Testing of a new antibody Preserved enzymatic activity Phosphorylation detection
Fresh Frozen Other Applications
Magnesium ATPase:
- Section of muscle incubated at 37’c @ pH values ….., ….. , ….
- Shows type.. …. …. .. .. . . . .. muscle fibres
Succinic dehydrogenase:
- section of ……
- Incubated with …….. …… at ……….
- NB the stain localises on mitochondria at sites of redox enzymes
Fresh Frozen Other Applications
Magnesium ATPase:
- Section of muscle incubated at 37’c @ pH values 9.2, 4.6, 4.2
- Shows type 1, type 11a, and 11b muscle fibres
Succinic dehydrogenase:
- section of muscle
- Incubated with succinate medium at 37’c
- NB the stain localises on mitochondria at sites of redox enzymes
Fresh Frozen tissue fixatives
Prior Cryo Microtome Cutting NO CHEMICAL FIXATIVE Sent ........ Sent in Michel’s transport .............. ...........
Type of fixation
Freezing with liquid …..
Freezing with ………
Freezing in ……… (-20C)
….. slides
Cut Slides Fixative solutions
Clarke’s ........... (70% Alcohol + ............. ............. Acid) 95% ......... 95% ........... Fo........ A................... Co........................... of above
Fresh Frozen tissue fixatives
Prior Cryo Microtome Cutting NO CHEMICAL FIXATIVE Sent fresh Sent in Michel’s transport medium O.C.T
Type of fixation
Freezing with liquid nitrogen
Freezing with C02
Freezing in cryostat (-20C)
adhesive slides
Cut Slides Fixative solutions
Clarke’s fixative (70% Alcohol + Glacial Acetic Acid) 95% Alcohol 95% Methanol Formalin Acetone Cocktail of above
Formalin Fixed Paraffin Embedded Tissue
Fixation - Pre-…………….
Sectioning - …………..
Storage - multiple years at room t…………………
Advantages - Preserves tissue ………..
Limitation - Over fixation can mask the ……….
Fresh frozen tissue
Fixation - Post - …..
Sectioning - …………
Storage - 1 year at -……c
Advantages - Preserves ……….. and antigen …………
Limitation - Formation of ice ……… may ………… affect tissue ……….
Formalin Fixed Paraffin Embedded Tissue
Fixation - Pre-embedding
Sectioning - Microtome
Storage - multiple years at room temperature
Advantages - Preserves tissue morphology
Limitation - Over fixation can mask the epitope
Fresh frozen tissue
Fixation - Post - sectioning
Sectioning - Crystat
Storage - 1 year at -80’c
Advantages - Preserves enzyme and antigen function
Limitation - Formation of ice crystals may negatively affect tissue structure
Rapid Freezing only
More ……………. retained
Cells appear …………..
Limited ………… detail
Formalin fixed Paraffin wax embedded
Less ………..
Wider ………….. spaces
Crisper ………… detail
Formalin fixed paraffin wax embedded
Less cytoplasm
Wider intercellular spaces
Crisper nuclear detail
Formalin fixed Paraffin wax embedded
Less cytoplasm
Wider intercellular spaces
Crisper Nuclear detail
Aim to freeze ……. - avoid ice ………. ………..
Provide …….. to tissue - ………… compound
Thin (4-10µ) sections - disposable ……..
Section adhesion to slides - brief/…… fix……… (20s- 1m)
Staining - ……. (reduced timings <5min)
Aim to freeze rapidly - avoid ice crystal damage
Provide support to tissue - embedding compound
Thin (4-10µ) sections - disposable blade
Section adhesion to slides - brief/light fixation (20s- 1m)
Staining - Rapid (reduced timings <5min)
Frozen sections - freezing
Require rapid fall in temperature –
avoids the formation of ……… ice crystals
Use CO2 cylinder – adiabatic expansion cools to -..oC
Use liquid nitrogen –temperature about -…….0C
Cool iso-pentane in liquid nitrogen ~ -……oC
Tissue immersed in OCT (Opt……. ………. Tempe………) embedding compound; cools efficiently as heat ………. away without
an ………….. layer of ……………..gas
Require rapid fall in temperature –
avoids the formation of large ice crystals
Use CO2 cylinder – adiabatic expansion cools to -30oC
Use liquid nitrogen –temperature about -1900C
Cool iso-pentane in liquid nitrogen ~ -150oC
Tissue immersed in OCT (Optimal Control Temperature) embedding compound; cools efficiently as heat conducts away without
an insulating layer of nitrogen gas
Frozen sections – rapid freezing with isopentane
Method A
Fresh sample in ….. on … foil
Supercooled i…………
Liquid nitrogen j………….
Method B
Fresh sample ori…………. in O…
Held on a r….. s……….
Suspend in isop……..
Method C
Fresh sample orientated in O…….
Placed in th…….e cr………… (-20C)
Leave to fre. (within 1-2 min)
Method A
Fresh sample in OCT on Al foil
Supercooled isopentane
Liquid nitrogen jacket
Method B
Fresh sample orientated in OCT
Held on a rigid support
Suspend in isopentane
Method C
Fresh sample orientated in OCT
Placed in the cryostate (-20C)
Leave to freeze (within 1-2 min)
