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CytoLyt® fixa+on and decalcifica+on
pretreatments alter an+genicity in normal +ssues compared to standard formalin
fixa+on
Penny Barnes, MD, FRCP(C) Capital Health District Health Authority and Dalhousie University, Halifax, NS
June 20, 2014
CytoLyt® fixa+on and decalcifica+on pretreatments alter an+genicity in normal +ssues compared to standard formalin
fixa+on
Jenne$e R. Gruchy, Penny J. Barnes, Kelly A. Dakin Haché
Applied Immunohistochemistry and Molecular Morphology, 2014. In press
• I have no conflicts of interest to disclose.
Learning objec+ves:
At the end of this session: • Par+cipants will be aware of the limita+ons of specific IHC an+bodies in the context of non-‐standard preanaly+c condi+ons, such as Cytolyt fixa+on for cytopathology specimens and decalcifica+on pretreatments.
• Par+cipants will be able to consider op+ons for valid external IHC controls for these specimens.
Outline
• Background • Study methods and results • Conclusions and future direc+ons
U+lity of immunohistochemistry
• In conjunc+on with accepted morphologic criteria, IHC is rou+nely used to detect specific cellular differen+a+on to aid in tumor classifica+on
• Prognos+c and predic+ve informa+on
IHC
• Increasing awareness of the importance of op+mizing and valida+ng IHC procedures
• Guideline documents have been published by interna+onal pathology organiza+ons
• The goal is to reduce the incidence of false nega+ve and false posi+ve IHC results, par+cularly in the context of predic+ve markers (Class II tests).
Am J Clin Pathol 2010;133:354-‐365.
Fixa+on
• Prior to processing, most +ssue specimens (biopsies, resec+ons) are fixed in formalin – The recommended fixaKve is 3.7 -‐ 4% w/v aqueous formaldehyde in phosphate buffer pH 7.4 nominal (range 7.2-‐7.6).
Alterna+ve fixa+ves and pretreatment procedures
• Non-‐gynecologic cytological specimens (fine needle aspira+ons, brushings, washings, fluids) in our lab are fixed using an methanol-‐based fixa+ve for liquid-‐based cytology: CytoLyt®
• Calcified specimens require decalcifica+on pretreatment using strong acid solu+ons.
Cytopathology specimens
• PosiKve controls for [fresh air-‐dried (or briefly fixed in ethanol or methanol or cytologic spray fixaKve)] cytologic preparaKons must be only fresh air-‐dried (or fixed in the same fixaKve) cytologic preparaKons derived from previously characterized paKent samples or cell lines.
Decalcifica+on
• Decalcified samples use separate controls which are fixed and decalcified in the same manner as the test.
• Separate validaKon procedures for each anKbody are performed.
Posi+ve controls
• PosiKve controls are valid only if they are fixed and prepared in the same manner as the Kssue samples that are tested in the assay.
• It is not only inappropriate to use posiKve controls that are processed differently from tested samples, but it may be diagnosKcally misleading.
External posi+ve and nega+ve controls
• Tissue arrays • Normal +ssues with predictable an+gen expression when feasible
• Tumor controls for some an+bodies e.g. Her2, ER, PR, Oct3/4
Accuracy of IHC in cytopathology cell blocks and decal specimens
• Is there a problem? • Are there specific an+bodies which are less robust in these sedngs?
• How can we develop our own external posi+ve and nega+ve controls?
Literature review
• There is no published research to date on the accuracy of IHC stains on CytoLyt® fixed cytological specimens compared with standard formalin fixed +ssue.
• Few reports on accuracy of IHC applied to +ssue pretreated with decalcifying agents
• Developed external control material for selected IHC an+bodies via brush prepara+on of tonsil, bronchial washing and serous fluid specimens
• LBC (SurePath®) • Mixed material, stained with green dye, applied one drop to IHC slide
Cytopathology 2011;4:243-‐6.
• An+bodies assessed: lymphocyte markers, vimen+n, EMA, p16, CK14, CK5/6, CK7, MN116, calre+nin, CA125
• Stored slides with external controls for 40 days on freezer • Reported expected staining pagerns for control material (macrophages, mesothelial cells, lymphocytes, bronchial cells, squamous epithelial cells)
• Proposed forma+on of other external control cocktails (aspirates of unfixed organs e.g. breast, thyroid, colon)
Cytopathology 2011;4:243-‐6.
• Limita+ons: – Difficult to iden+fy cell types of cytology material obscured by IHC stain
– Difficult to iden+fy low expressing cells without intact architecture
• Documenta+on of procedure and +ssue controls seems less stringent in the cytopathologic literature
• Reviewed literature for 9 years on IHC in cytopathology
• Only 11/87 papers (13%) described use of posi+ve and nega+ve controls on iden+cally prepared specimens
Diagn Cytopathol. 2011;4:245-‐50.
Histochemical Journal 1984;16:771-‐787.
Decalcifica+on studies
• Tissues pretreated with a number of decalcifying agents exhibited variable preserva+on of histological structure and an+genic reac+vity.
• Harsher decalcifying agents (HCl, HNO3) showed reduced sensi+vity.
• Newer an+bodies and techniques have not been evaluated systema+cally.
Study Objec+ves
• To systema+cally compare the expression of commonly used an+bodies in normal +ssues exposed to CytoLyt® or decalcifying agents compared to standard formalin fixed +ssues.
• To develop external control material for Cytopathology and decalcifica+on procedures
Methods
• CDHA Research Ethics Board approval
• Grant funding from the Capital District Health Authority Trainee Research Fund.
• Assembled bogles with labels, 4 solu+ons, created collec+on and results worksheet
Methods
• Prospec+vely collected 10 of each +ssue type: kidney, breast, skin, appendix, lung – monitored OR list – directly called surgeons/OR staff: “SEND FRESH!” – on arrival, JRG was called to the gross room – collected relevant data (i.e. pt name, surgical number, surgeon, date, & +me)
Methods
– Cases excluded if no normal +ssue could be sampled without interference of usual grossing protocol
– 1 por+on of +ssue sampled (0.3cm x 1cm x 0.5cm), divided into 4 pieces
– Recorded study # on requisi+on – Specimen then placed in fresh formalin for regular gross handling and processing
Methods • a piece of each study +ssue sample went into 1 of 4 solu+ons
Standard formalin
CytoLyt® Decal (Formic acid)
Rapid decal (Hydrochloric acid)
Decalcifying agents
• Leica Decalcifier I® -‐ 10% formic acid (bogle C) • Leica Decalcifier II® -‐ 5% hydrochloric acid (bogle D).
Methods
• ….fixa+on +me was controlled
4 pieces of fresh +ssue
3 in formalin ≥ 24 hrs
‘A’ specimen processed and embedded
1 transferred to ‘C’ (decal)
~8hrs
rinsed with water x 15
mins
‘C’ specimen processed and embedded
1 transferred to ‘D’ (rapid decal) ~4hrs
rinsed with water x 15
mins
‘D’ specimen processed and embedded
1 in ‘B’ (Cytolyte) ≥ 24 hrs
‘B’ specimen processed and embedded
Collec+on Worksheet
Methods
• Normal +ssue samples con+nued through rou+ne +ssue processing under RS-‐301-‐#
• A panel of IHC was completed on 4 µm sec+ons of paraffin embedded surgical material
• Automated immunostainer (Ventana Benchmark XT)
ANTIBODY CLONE SPECIES SUPPLIER DILUTION ANTIGEN RETRIEVAL DETECTION
AE1/AE3 AE1:AE3 monoclonal mouse Dako 1/100 CC1 (30min) Ultraview
CK5/6 34BE12 monoclonal mouse Ventana Predilute CC1 (30min) Iview
CK8/18 B22.1 & B23.1 monoclonal mouse Cell Marque 1/100 Protease (4min) Iview
CK7 OV-TL12/30 monoclonal mouse Dako Predilute CC1 (8min) Iview
CK20 K20.8 monoclonal mouse Dako 1/100 Protease (4min) Iview
P63 BC4A4 monoclonal mouse Biocare Marque Predilute CC1 (30min) Iview
TTF-1 8G7G3/1 monoclonal mouse Dako 1/100 CC1 (30min) Iview
ER SP1 rabbit monoclonal Ventana Predilute CC1 (30min) Ultraview
CEA TF 3H8-1 monoclonal mouse Ventana Predilute None Iview
VIMENTIN Vim3B4 monoclonal mouse Ventana Predilute CC1 (60min) Ultraview
S100 protein Polyclonal polyclonal rabbit Dako 1/3000 CC1 (8min) Iview
D2-40 D2-40 monoclonal mouse Dako Predilute CC1 (8min) Iview
LCA RP2/18 monoclonal mouse Ventana Predilute None Iview
CD3 2GV6 monoclonal mouse Ventana Predilute CC1 (30min) Iview
CD20 L26 monoclonal mouse Ventana Predilute CC1 (8min) Iview
CALRETININ Polyclonal polyclonal rabbit Invitrogen 1/50 CC1 (30min) Iview
CHROMOGRANIN LK2H10 monoclonal mouse Ventana Predilute CC1 (30min) Iview
SYNAPTOPHYSIN 27G12 monoclonal mouse Novocastra 1/25 CC1 (30min) Iview
Methods
NORMAL TISSUE STAIN
LUNG TTF-‐1, CK7
SKIN p63, CK5/6
BREAST ER
KIDNEY CK8/18, AE1/AE3
APPENDIX CD3, CD20, CK20, D2-‐40, LCA, S100, Calre+nin, Synaptophysin, Chromogranin A, Vimen+n
Methods
• Two inves+gators (KDH and PJB), interpreted the IHC expression pagerns semi-‐quan+ta+vely in a blinded manner
• Assessed: • expression pagern (nuclear, cytoplasmic, membranous) • percentage of posi+ve cells • intensity • distribu+on • background/non-‐specific staining
• Data was inserted into a secure database by JRG, SPSS v.19
Results • Expected expression of all an+bodies was seen in +ssues fixed with standard formalin
• Several an+bodies were very robust, showing op+mal expression across all 4 fixa+ves and pretreatments
• Some an+bodies showed reduced sensi+vity and others showed complete absence of expression with non-‐standard fixa+ve/pretreatment
FORMALIN CYTOLYT® FORMIC ACID HYDROCHLORIC ACID
AE1/AE3 CK 5/6 CK 8/18 CK7 CK20 P63 TTF1 ER CEA VIMENTIN S100 D2-40 LCA CD3 CD20 CALRETININ CHROMOGRANIN A SYNAPTOPHYSIN
Summary of IHC results for each pretreatment group
Black – optimal or near optimal expression Grey – reduced expression White – absent or near absent expression
Robust An+bodies (4/17)
• AE1/AE3 • Vimen+n • CK20 • CEA
AE1/AE3
• kidney • op+mal expression
– cytoplasmic – strong diffuse expression in distal tubules & Bowman’s capsule; weaker expression in proximal tubules
Formalin CytoLyt ®
Formic Acid HCl
AE1/AE3
Vimen+n
• appendix • op+mal expression
– cytoplasmic – lymphocytes (cytoplasm), endothelial cells and peripheral nerves
Formalin CytoLyt ®
Formic Acid HCl
Vimen+n
Results
• Altered expression was observed with several an+bodies compared to standard formalin fixa+on
CytoLyt® fixa+on • Absent or near absent expression of:
– thyroid transcrip+on factor 1 (TTF-‐1) – D2-‐40 – CD20
• Reduced expression of : – p63 – estrogen receptor (ER) – S100 protein – CD3 – calre+nin – chromogranin A – synaptophysin
TTF-‐1
• clone 8G7G3/1 • lung • op+mal expression
– nuclear – strong in terminal bronchiolar epithelium – strong in type 2 pneumocytes
Formalin
CytoLyt ®
TTF-‐1
CD20
• appendix • op+mal expression
– membranous – all B cells, including germinal center B cells
Formalin
CytoLyt ®
CD20
D2-‐40
• appendix • op+mal expression
– cytoplasmic, membranous (mesothelial cells) – lympha+c endothelial cells – Cajal cells
Formalin
CytoLyt ®
D2-‐40
p63
• skin • op+mal expression
– nuclear – epidermis
Formalin
CytoLyt ®
p63
S100 protein
• appendix • op+mal expression
– nuclear and cytoplasmic – peripheral nerve, ganglion cells – fat cells
Formalin
CytoLyt ®
S100
Decalcifying agents: 10% formic acid
• Exposure to formic acid for 8 hours had less impact with reduced expression observed for only three an+bodies: – TTF-‐1 – CK7 – CK8/18
Formalin
Formic Acid
TTF-‐1
Formalin
Formic Acid
CK7
Decalcifying agents: 5% hydrochloric acid (4 hours)
• Absent or near absent expression of : – TTF-‐1
• Reduced expression of: – CK5/6 – CK7 – p63 – ER – LCA – CD3 – CD20 – synaptophysin
Formalin
HCl
TTF-‐1
Formalin
HCl
CK7
Formalin
HCl
p63
ER
• breast • op+mal expression
– nuclear – breast epithelial cells
Formalin
HCl
ER
LCA
• appendix • op+mal expression
– membranous – lymphocytes
Formalin
HCl
LCA
Summary of Results CytoLyt® -‐ complete or near complete loss of expression of:
-‐ TTF-‐1, D2-‐40, CD20 -‐ reduced expression of:
-‐ p63, ER, S100 protein, CD3, calre+nin, chromogranin A, synaptophysin
Decal (10% formic acid, 8 hrs) -‐ reduced expression of :
-‐ CK7, CK8/18, and TTF-‐1 Rapid decal (5% hydrochloric acid, 4 hrs) -‐ complete loss of TTF-‐1 -‐ reduced expression of:
– ER, p63, LCA, CK7, CK5/6, CD3, CD20, synaptophysin
Limita+ons of our study
• Sample size • We have controlled for the fixa+ve type, but not the liquid based cytopathology processing for CytoLyt® fixed samples and cell blocks
• Select panel of IHC an+bodies/clones/protocols studied, +me exposed to decal acids
• Low and high expressing +ssues (e.g for ER) not assessed
Next steps
• Proper external controls need to be developed – microarrays of normal +ssues handled iden+cally as test specimens (CytoLyt ; formalin/formic acid)
– op+mize protocols – revalidate protocols – on-‐slide controls
Next steps
• It is possible that with some fixa+ve/pretreatment condi+ons, some an+gens cannot be iden+fied, despite agempts at op+miza+on.
Conclusion
• Pathologists must be aware of the limita+ons of specific IHC an+bodies in the context of non-‐standard fixa+on and decalcifica+on pretreatments.
References
Torlakovic EE, Riddell R, Banerjee D et al. CAP-‐ACP Best prac+ce recommenda+ons for standardiza+on of immunohistochemical tests. Am J Clin Pathol 2010;133:354-‐365.
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Hansen, T, Pedersen, H, Brauner, V, et al. Control specimens for immunocytochemistry in liqid-‐based cytology. Cytopathology. 2010; 1-‐4.
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100 Journal Ar+cles. DiagnosKc Cytopathology. 2010; 39, 245-‐250.
MCQ: Regarding use of IHC on cytopathology cell block prepara+ons:
A. Posi+ve controls used for histopathology are valid.
B. Liquid-‐based cytopathology technology employs formalin fixa+on.
C. Posi+ve controls should be fixed and prepared in the same manner as cytopathology specimens.
D. All commonly used IHC an+bodies are robust and reliable in cytopathology specimens.
MCQ: Regarding use of IHC on cytopathology cell block prepara+ons:
A. Posi+ve controls used for histopathology are valid.
B. Liquid-‐based cytopathology technology employs formalin fixa+on.
C. Posi+ve controls should be fixed and prepared in the same manner as cytopathology specimens.
D. All commonly used IHC an+bodies are robust and reliable in cytopathology specimens.
