Jaffe HP IMMUNOHISTOCHEMISTRY (Ch. 3) Flashcards
Underlying principle of IHC?
Antigen-antibody reaction
What does success of the antibody-antigen reaction depend on?
Antigenic epitopes recognized by diagnostic antibody must maintain reactive conformation.
Why is rapid fixation important in terms of IHC?
Enzymatic degradation begins immediately after biopsy/resection, causing conformational changes in epitopes.(Note that additional changes will also happen as a result of fixation!)
Why might fixation interfere with antigenicity?
- Causes conformational changes in antigen epitopes. 2. Chemically modifies epitopes.
Advantages of neutral buffered formalin?
Inexpensive. sterilizing properties. Good preservation of morphology.
How does formalin fix tissues?
Crosslinks aldehydes in proteins. Note that epitopes can be grouped as formalin-resistant, highly formalin-sensitive, or having a time-dependent sensitivity to formalin.
How can antigenicity be retrieved after formalin fixation?
- Old way: proteolytic enzymes
- New way: heat-induced epitope retrieval (HIER)
What are the basic steps of HIER?
- Fix tissue
- Heat to 100 decrees C for up to 30 mins
Presumably hydrolytic cleavage of formaldehyde crosslinks, inner epitope unfolding, extraction of calcium ions
What are the 2 major categories of primary antibodies in IHC?
- polyclonal
- monoclonal
Difference between polyclonal and monoclonal antibodies?
polyclonal- made by harvesting serum (antibodies) from an animal injected with antibody. Less specific because contains multiple antibodies that react to multiple epitopes. Can’t standardize as immune responses differ over time and between animals.
monoclonal- product of a single immortalized antibody-producing cell. Based on hybridoma technology where a single antibody-producing mouse cell was fused with a mouse plasmacytoma cell line. hybrid cells are then clonally expanded. Known composition and reactivity.
What are 2 types of IHC positive controls?
- On slide tissue that contains Ag of interest
- Internal control- if the tissue to be tested has cells that normally express Ag of interest
What is a detection system in IHC?
enzyme, chromogenic substrate, and link or bridge reagent that brings enzyme and primary Ab into proximity
What types of detection systems exist in IHC?
polymer-based
CARD (catalyzed reporter deposition ) or CSA (catalyzed system amplification) method
avidin biotin immunoperoxidase complex (ABC)- problematic because there is high endogenous biotin and hence high background staining in some tissues
What issue is likely to arise if steps are not taken to block biotin?
False positives due to endogenous biotin reactivity
name 3 fixatives used for marrow biopsies
NBF
Zenker’s
B5
AZF (acid zinc formalin)- best in terms of morphology and nuclear detail
What are some factors that can help you to differentiate background staining from true staining in IHC?
- Location of staining (nuclear, cytoplasmic, membranous)
- Intensity of staining
- Comparison with controls
Recommended IHC for small B-cell lymphomas?
CD20, CD79a, CD3, CD5, CD10, CD23, CD21, BCL2, BCL6, cyclin D1, IgD, kappa and lambda light chains, Ki-67, LEF1, MUM1, SOX11
Recommended IHC for aggressive B-cell lymphomas?
CD20, CD3, CD5, BCL2, BCL6, CD10, IRF4/MUM1, MYC, EBER ISH
Recommended IHC for plasma cell and plasmablastic neoplasms.
CD20, CD79a, CD3, kappa and lambda light chains, Ig heavy chains, CD56, CD138, MUM1, ALK, EMA, EBER ISH, HHV8
Recommended IHC for CHL?
CD20, CD79a, CD3, kappa and lambda light chains, Ig heavy chains, CD56, CD138, MUM1, ALK, EMA, EBER ISH, HHV8
Recommended IHC for NLPHL?
CD20, CD3, IgD, OCT-2, BCL6, CD21, PD-1
Recommended IHC for extranodal PTCL?
CD20, CD3, CD5, CD4, CD8, CD2, CD7, CD25, CD30, CD56, TIA-1, granzyme B, β-F1, ALK, TCR delta, EBER ISH
Recommended IHC for nodal PTCL?
CD20, CD3, IgD, OCT-2, BCL6, CD21, PD-1
What are some reasons why certain stains are targeted for IHC in hemepath?
Lineage markers
Maturity markers/ blast markers
Markers of altered gene products
Proliferation markers (e.g. to help distinguish between neoplastic and reactive germinal centers)
Oncogene associated products (e.g. p53 IHC for TP53 mutation)
Translocation-associated markers – e.g. Overexpression of cyclin D1 as a result of the t(11;14)(q13;q34) in MCL, ALK-NPM1 translocation in ALCL
Rearrangement associated markers e.g.c-MYC
BRAF v600E mutation in HCL, ECD, and LCH
What lymphomas characteristically show PD-L1 amplifications?
Hodgkin lymphoma, primary mediastinal large B-cell lymphoma
What IHC panel would you order to distinguish between AML and lymphoblastic leukemia?
CD34, TdT, MPO, CD33, CD68 (KP-1 and PGM-1), glycophorin A, CD61, CD20, CD79a, PAX5, CD3, and CD1a + others as necessary
Monocytic markers by IHC?
CD11c, CD14, CD64, CD4, CD163, and lysozyme
Characteristic immunophenotype of APL?
MPO, CD13, and CD33
Negative for HLA-DR, neg to weak CD34
Possible aberrant CD2 and CD56 in microgranular variant.
Histiocytic/dendritic markers?
CD68, CD163, CD11c, CD14, lysozyme, CD4 and PU.1.
BPDCN Markers?
CD4, CD43, CD56, CD123, TCL-1/TCF4
Mastocytosis markers?
CD117, CD25 (aberrant), tryptase
LCH Markers
CD68, CD163, CD11c, CD14, lysozyme, CD4 and PU.1.
Role of IHC in hemepath?
Diagnostics
Prognostic information
Predictive information
Helps therapeutic decision making
What is rituximab?
Unconjugated (“naked”)Anti-CD20 MAb
What is Brentuxumab vedotin?
Anti-CD30
What is alemtuzumab?
Anti-CD52
Gliteritinib?
FLT3 inhibitor
Gemtuzumab?
Anti CD33
Daratumumab?
Anti-CD138
Inotuzumab ozogamicin?
Anti-CD22
Blinotumomab
Anti-CD19
Basiliximab and Daclizumab?
Anti-CD25. Used to prevent GVHD/graft rejection
Vemurafenib
BRAF Inhibitor
IHC vs ISH?
both interrogate targets in situ—that is, on frozen or paraffin-embedded tissue sections—and they have similar detection systems.
Differences: target type and chemistry of identification of target
IHC- protein; FFPE tissue only
ISH: DNA or RNA; frozen section, FFPE, cell suspensions, cytogenetic preparations
Reactivity (hybridization) is based on complementarity between the sequence of interest and the designed probe, rather than on antigen-antibody recognition.
When might you opt for ISH over IHC?
Antibodies for IHC unavailable
IHC has issues with high background staining (e.g. kappa and lambda for B-cell clonality)
Proteins are rapidly secreted by cells of interest
DNA/RNA content>protein
Infectious agents- EBV
CISH for EBER: Explain how it works.
Targets = EBV-encoded RNAs (EBERs), which are short nuclear transcripts that are present early in latent infection and in high copy number (approximately 10 copies/cell).
EBER CISH is performed on formalin-fixed, paraffin-embedded tissue sections and are preferable to the commonly used IHC target
Alternative is IHC for latent membrane protein (LMP)
Note: always ensure that intact RNA is detectable in the tissue of interest using the polyuridine probe against the poly A tail of mRNA (“RNA control”).
What are some factors that could contribute to poor IHC results?
Old slide (unstained slides lose antigenicity over time)
Allowing slide to dry may cause background staining
Incorrect pH for Ag retrieval
Incorrect temperature in HIER
DDx for elderly pancytopenic patient
MDS, HCL, SMXL, T-negative LGL leukemia, HLH
