CytoLyt fixa+on and decalcifica+on pretreatments alter an+genicity in normal +ssues compared to standard formalin fixa+on

Transcription

1 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

2 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, In press

3 I have no conflicts of interest to disclose.

4 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.

5 Outline Background Study methods and results Conclusions and future direc+ons

6 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

7 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).

8 Am J Clin Pathol 2010;133:

9 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 ).

10 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.

11 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.

12 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.

13 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.

14 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

15 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?

16 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

17 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.

18 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.

19 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

20 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:

21 Histochemical Journal 1984;16:

22 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.

23 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

24 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

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26 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)

27 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

28 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)

29 Decalcifying agents Leica Decalcifier I - 10% formic acid (bogle C) Leica Decalcifier II - 5% hydrochloric acid (bogle D).

30 Methods.fixa+on +me was controlled

31 4 pieces of fresh +ssue 3 in formalin 24 hrs 1 in B (Cytolyte) 24 hrs A specimen processed and embedded 1 transferred to C (decal) ~8hrs 1 transferred to D (rapid decal) ~4hrs B specimen processed and embedded rinsed with water x 15 mins rinsed with water x 15 mins C specimen processed and embedded D specimen processed and embedded

32 Collec+on Worksheet

33 Methods Normal +ssue samples con+nued through rou+ne +ssue processing under RS # A panel of IHC was completed on 4 µm sec+ons of paraffin embedded surgical material Automated immunostainer (Ventana Benchmark XT)

34 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

35 Methods NORMAL TISSUE LUNG SKIN BREAST KIDNEY APPENDIX TTF- 1, CK7 p63, CK5/6 ER STAIN CK8/18, AE1/AE3 CD3, CD20, CK20, D2-40, LCA, S100, Calre+nin, Synaptophysin, Chromogranin A, Vimen+n

36 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

37 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

38 Summary of IHC results for each pretreatment group 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 FORMALIN CYTOLYT FORMIC ACID HYDROCHLORIC ACID Black optimal or near optimal expression Grey reduced expression White absent or near absent expression

39 Robust An+bodies (4/17) AE1/AE3 Vimen+n CK20 CEA

40 AE1/AE3 kidney op+mal expression cytoplasmic strong diffuse expression in distal tubules & Bowman s capsule; weaker expression in proximal tubules

41 AE1/AE3 Formalin CytoLyt Formic Acid HCl

42 Vimen+n appendix op+mal expression cytoplasmic lymphocytes (cytoplasm), endothelial cells and peripheral nerves

43 Vimen+n Formalin CytoLyt Formic Acid HCl

44 Results Altered expression was observed with several an+bodies compared to standard formalin fixa+on

45 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

46 TTF- 1 clone 8G7G3/1 lung op+mal expression nuclear strong in terminal bronchiolar epithelium strong in type 2 pneumocytes

47 TTF- 1 Formalin CytoLyt

48 CD20 appendix op+mal expression membranous all B cells, including germinal center B cells

49 CD20 Formalin CytoLyt

50 D2-40 appendix op+mal expression cytoplasmic, membranous (mesothelial cells) lympha+c endothelial cells Cajal cells

51 D2-40 Formalin CytoLyt

52 p63 skin op+mal expression nuclear epidermis

53 p63 Formalin CytoLyt

54 S100 protein appendix op+mal expression nuclear and cytoplasmic peripheral nerve, ganglion cells fat cells

55 S100 Formalin CytoLyt

56 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

57 TTF- 1 Formalin Formic Acid

58 CK7 Formalin Formic Acid

59 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

60 TTF- 1 Formalin HCl

61 CK7 Formalin HCl

62 p63 Formalin HCl

63 ER breast op+mal expression nuclear breast epithelial cells

64 ER Formalin HCl

65 LCA appendix op+mal expression membranous lymphocytes

66 LCA Formalin HCl

67 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

68 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

69 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

70 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.

71 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.

72 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: Leong, T, Cooper, K, Leong A, et al. Immunohistology- Past, Present, and Future. Adv Anat Pathol. 2010; 17(6): Idikio, HA. Immunohistochemistry in diagnos+c surgical pathology: contribu+ons of protein le{- cycle, use of evidence- based methods and data normaliza+on on interpreta+on of immunohistochemical stains. Int J Clin Exp Pathol. 2010; 3(2): Hansen, T, Pedersen, H, Brauner, V, et al. Control specimens for immunocytochemistry in liqid- based cytology. Cytopathology. 2010; 1-4. Goldstein, NS, Hewig, SW, Taylor, CR, et al. Recommenda+ons for improved standardiza+on of immunohistochemistry. Appl immunohistochem Mol Morphol. 2007;15(2): Grizzle,WE. Special symposium: fixa+on and +ssue processing models. Biotechnic & Histochemistry. 2009;84(5): Haack, L, Shalkham, J. Valida+on in the Cytopathology Laboratory: Its Time Has Come. Cytotechnology Forum. 2007; 35(8): Essen, HF, Verdaasdonk, MA, Elshof, SM, et al. Alcohol based +ssue fixa+ve as an alterna+ve for formaldehyde: influence on immunohistochemistry. Journal of Clinical Pathology. 2010;63: Athanasou, NA, Quinn, J, Heryet, A, et al. Effect of decaldifica+on agents on immunoreac+vity of cellular an+gens. J Clin Pathol. 1987;40: Maghews, J, Mason, G. Influence of decalcifying agents on immunoreac+vity of formalin- fixed, paraffin- embedded +ssue. Histochemical Journal. 1984;16: Noraidah, M, Ghoddoosi, M, et al. RCL2, a poten+al formalin subs+tute for +ssue fixa+on in rou+ne pathologic specimens. Histopathology. 2012; 60, Colasacco, C, Mount, S. Documenta+on of Immunocytochemistry Controls in the Cytopathologic Literature: A Meta- Analysis of 100 Journal Ar+cles. DiagnosKc Cytopathology. 2010; 39,

73 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.

74 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.

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