Possible pit falls at each step in IHC

stainingHazel Chambers-Smith,

Sri Lanka Workshop of Basics of Immunohistochemistry, June 2019

Pitfalls in IHC

• There are many steps involved in

successful processing of tissue

• There are further steps involved in

successful IHC staining!

• When we are fully automated, we take

for granted the numerous steps involved

in producing high quality IHC slides

A recap of what IHC is

Factors affecting an IHC result

1.Tissue handling and fixation

• Avoid ischemia of tissues

• This leads to DNA/RNA degradation

• Can also see activation of tissue enzymes

and autolysis

• Prompt fixation

• 10% neutral buffered formalin for 10-24

hours

• Some antibodies don’t work using formalin;

frozen sections fixed in acetone may be

required (becoming very uncommon)

• Beware of over fixation

• Acetone fixation can cause poor

morphology but with good epitope

retention

• Soluble antigens may be diffused out

during processing

• Processing of tissue must also be good!

• Observe appropriate tissue sampling

recommendations – tissue should be

smaller than the cassette!

Poor fixation effects in IHC

• Doesn’t always uniformly affect panels

• Some antibodies are more sensitive

than others

• HE morphology can look fine so it is

misleading

Fixation effects MSI

MSH6

PMS2

MLH1

2. Sectioning and storage

• We routinely cut sections at 3-4µm

• Use a good quality adhesive slide

• Superfrost plus, Dako flex, Matsunami

slides are manufacturer recommended

(our experience is these are good)

• “Home-made” adhesive slides must be

QC’d after each batch to ensure quality

and to reduce repeats due to “float-off”

• Cut paraffin sections should be

protected from degradation by coating

with paraffin or vacuum storage,

keeping cool – ensure all water is

removed from the tissue sections first

• Thicker sections can show higher

background staining

• Stick to “tried and tested” methods

• The size of the section may affect

staining

• Each slide gets 100ul of antibody to

cover the section and control

• Sections should be centrally placed

away from the IHC label and not

overhanging the edges

• Consider not using the whole block for

the IHC slide

• A photocopy of the slide marked with

the target area can be used (how IHC is

done on megablocks-breast/prostate)

• Drying-Extended drying at 60 degrees is

proven to destroy antigenicity (1+ ER

cases could be potentially affected)

3.Antigen and epitope retrieval• Fixation masks

epitopes

• Not necessary for

frozen sections/cyto

• Heat induced epitope

retrieval (HIER) is

common

• Autoclave

• Microwave

• Waterbath

• Pressure cooker

• Instrumentation

The right retrieval • Important that the right

solution is used

• One antibody can

behave very differently

when the wrong

retrieval solution is

used

• For example,

• a low pH solution would

not show staining for

antibody C

• A neutral pH would not

stain antibody B

• Enzymes can be used for antigen

retrieval but do not “overcook”

• Microwave boiling can cause

mechanical lifting of sections

• Remember HIER and enzymes can be

used

• Some antigens need no retrieval

• Use good quality dH20 for reagents

4. Antibodies

• Use high affinity antibodies

• Use a well known antibody with documented

literature citations for patient diagnostics

• Use negative controls to check for

background/non-specific staining

• Store antibodies correctly and use aseptic

techniques during aliquoting and working

dilution preparations – can cause problems in

antibody behaviours and staining intensity

Manual IHC-Antibodies

• Ensure working dilution is made up

correctly

• Use a timer in manual methods!

• Don’t let the slide dry out

• Consider dilution of antibody and

incubation overnight at 4°C if time is

running out

• Diluents can affect antibody

performance

RTU/prep kit dispenser issues

• Dispenser nozzles prone to

drying up

• PPT clogs up the hole

preventing reagents being

dispensed

• Amplification kits very

susceptible

• Leading cause of failed PMS2

(4.5 hr protocol!!)

• Replacing caps and frequently

checking dispensers will

reduce fails in the lab.

5. Secondary antibodies

• Ensure the antibody

is appropriate for the

primary

• Ensure you have a

chromogen-linked

tertiary (detection)

antibody appropriate

for the secondary

• If using a mouse monoclonal, consider

a rabbit-anti-mouse secondary

• Then use a labelled Goat-anti-rabbit

linked to a chromogen

• Optiview DAB:

6. Detection systems

• Avoid biotin detection systems in

spleen, brain, liver and kidney tissues

are they are high in endogenous biotins

• Avoid alkaline phosphatase detection

systems in tissues rich in endogenous

peroxidases; bone marrow and

lymphoid tissue

• Synthetic polymer/multimer detection

systems now common (eg Optiview)

• Highly specific, clean, very sensitive

• Configuration of molecule avoids “steric

hindrance”

• Choose an appropriate chromogen

• DAB might not be appropriate for

pigmented lesions

• AEC (red/soluble in alcohol) might not be

appropriate due to its solubility

7. Post IHC staining issues

• Heavy counterstains can make

interpretation difficult (use automation or

“on-board” Hx).

• Soaking (soluble) chromogens in

alcohol and losing staining

• Ability for your team to appropriately

quality check staining (don’t just look for

brown)

• E.g “Why is the signal nuclear when it

should be membraneous”…

Experience is the best skill to identify

errors

• Product catalogues

are an amazing

resource to help with

quality checking

stains

• Cell marque

• Biocare

• Dako

• Leica

• Most contain

differential dx panels

• Great teaching and

learning resource

• Experienced colleagues are not always

available

• An in house developed interpretation

guide is a good resource and specific to

in house controls

In-house interpretation guide

8. Platform choice

• Having a single platform may restrict

labs to a slightly compromised service

• No one instrument can stain every

antibody available perfectly

• Having a second platform increases

flexibility but it is more difficult to

manage reagents etc

Platform choice

• Main vendors (Roche, Leica, Dako) all

have proprietary reagents in detection

kits which restricts flexibility, but the

results are usually more robust and

reliable.

• “Locked down” methods increasingly

employed in theranostic IHC eg

ALK/PDL1 testing

Summary

• Fixation

• Slide preparation + controls

• Retrieval method

• Antibody

• Detection system

• Counterstaining/mounting

• QC checking

• Interpretation

Check out:

• https://www.nordiqc.org/

• https://ukneqas.org.uk/

Any questions?