2nd NordiQC Conference on Applied Immunohistochemistry

Content

IHC and process validation

an aid towards standardization ?

▪ Background

▪ Definitions

▪ Process validation model

▪ Practical example

Content

▪ Background

▪ Definitions

▪ Process validation model

▪ Practical example

Background : lab challenges today

▪ Higher volume vs lower staffing

▪ More direct link to patient treatment

▪ Less patient material available

▪ Faster turn arround times (TAT)

▪ Lab specific settings vs standardization

▪ Quality management

▪ Higher demands from inspecting agencies

Background : “The Belgian situation”

Fed. Dept. of Health

▪ = official recognition of all pathology lab’s in Belgium

▪ Mandatory for every lab

▪ Royal Decree : 2011

▪ Practice guideline : 2014

Fed. Dept. of Economics

▪ ISO accreditation

▪ BELAC

▪ Not mandatory

▪ Specific tests within a scope

▪ ISO 15189

National accreditation programs moving to ISO 15189

Background : Practice guideline – ISO 15189

Technical Requirements

Personnel

Accommodation and environmental conditions

Laboratory equipment, reagents, and consumables

Pre-examination processes

Examination processes

Post-examination processes

Reporting & release of results

Laboratory information management

Pre-analytical

Analytical Post -

analytical

Management requirements Men Machine Means Methods

Laboratory Information System

Practice guide – ISO 15189

IHC : multistep & complex process

IHC Staining

Pre-analytical

Analytical Post -

analytical

Pre-analytical

Analytical Post -

analytical

Laboratoy Information System

Men Machine Means Methods

IHC Staining Tissue Fixation Preparation Sectioning Pre-treatment

Interpretation Control Cut-off level Reporting

Primary Ab Visualisation Staining platform

Process model Similar approach

IHC : from staining method to total process ?

IHC Staining

Pre-analytical

Analytical Post -

analytical Laboratoy Information System

Men : labtech’s, pathologists,…

Machine : processing, cutting, staining,…

Means : reagents, consumables, storage,…

Methods : fixation, processing, staining,…

IHC Staining Tissue Fixation Preparation Sectioning Pre-treatment

Interpretation Control Cut-off level Reporting

Primary Ab Visualisation Staining platform

Available info ?

▪ Validation of instruments

▪ Optimalization of methods

▪ Validation of methods

▪ IQC & EQC

▪ Reporting

▪ Etc …

▪ What about critical steps and/or influencing factors ? ▪ Known ? ▪ Effect ? ▪ Control ? ▪ Standardization ? ▪ Validation ?

▪ Total process ?

▪ Added value ?

IHC : from staining method to total process ?

IHC : multistep & complex process - Questions

▪ Do we have confidence in our IHC process ?

▪ Is it capable of consistently delivering quality results ?

▪ Can we demonstrate that the process works as intended ?

▪ Can we detect a process drift ?

▪ Do we know the critical attributes throughout the process ?

▪ How many steps / influencing factors are controlled ?

IHC : multistep & complex process - Questions

▪ Confidence in process Failure rate ?

▪ Consistent process Standardization

▪ Process works as intended Meets acceptance criteria

▪ Critical attributes known What are the risk factors ?

▪ Influencing factors identified

Prospective risk analysis = process validation

Process validation : new ?

▪ Process validation = Prospective risk analysis

▪ Used in pharma (GxP), clinical biology, industry, …

▪ Risk management integrated in quality management (Practice guidelines & ISO 15189)

▪ Different methods available

▪ Uses available information

▪ “Lab” tailored

Drivers for process validation

▪ Reduction of error through risk management and continual improvement

▪ Reduction of risk levels through risk reducing measures

▪ Higher degree of confidence in process

▪ Higher degree of standardization

▪ Higher degree of quality

Process validation = Framework for “in house” standardization

Content

▪ Background

▪ Definitions

▪ Process validation model

▪ Practical example

“Establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting it’s pre-determined specifications and quality attributes”

(FDA)

Definitions : VALIDATION

“Establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting it’s pre-determined specifications and quality attributes”

documented evidence

high degree of assurance

pre-determined specifications and quality attributes

▪ documentation

▪ confidence in the process

▪ validation criteria

Definitions : VALIDATION

“A series of actions or steps taken in order to achieve a particular end.”

▪Proces : – Manufacturer – User

▪ Integration / process design

Definitions : PROCESS

“Process validation is defined as the collection and evaluation of data, which establishes (scientific) evidence that a process is capable of consistently delivering quality products.”

Definitions : PROCESS VALIDATION

Definitions : PROCESS VALIDATION

Type of process validation Principle

Prospective/progressive risk analysis on total process

Concurrent during normal “production”, 3 batches monitored

Retrospective after process, using controls

Revalidation change in process

Types of process validation :

Prospective risk analysis

“Risk is the likelihood that there is damage, taking into account the extent of the damage.”

- or -

“Risk is the combination of the probability of occurrence of harm and the severity of that harm.”

(ISO/IEC Guide 51).

(≠ Hazard = “a potential source of harm”)

Definitions : RISK

Content

▪ Background

▪ Definitions

▪ Process validation model

▪ Practical example

Process validation method : steps

▪ Process validation = prospective risk analysis

▪ Steps : 1. Define scope for process validation 2. Describe process 3. Determine risk criteria 4. Perform risk assessment 5. Determine validation criteria 6. Validate appropriate risk factors/acceptance criteria 7. Maintain in a validated state

1. Define the scope for validation

IHC Staining

Pre-analytical

Analytical Post -

analytical

Interpretation Control Cut-off level Reporting

Tissue Fixation Preparation Sectioning Pre-treatment

Primary Ab Visualisation Staining platform

IHC Staining SAMPLE – REQUEST FOR STAINING

REPORT

1. Define the scope for validation

IHC Staining

Pre-analytical

Analytical Post -

analytical

Interpretation Control Cut-off level Reporting

Tissue Fixation Preparation Sectioning Pre-treatment

Primary Ab Visualisation Staining platform

IHC Staining process

Confirmation testing Correlation IHC - ISH

• Reagent storage • Reagent acceptance testing

• Instrument maintenance • Instrument acceptance

testing

• Time to fixation • Transport monitoring

• Processing

• Control preparation • Control storage

• Histotech - pathologist • Labmanagement • Quality management

• Lab. Information System

SAMPLE – REQUEST FOR TESTING

REPORT & FOLLOW UP OF RESULTS

1. Define the scope for validation

IHC - Single stain/panel IHC - Double stain

Kappa/ Lambda doublestain BioCare Medical K/L prim. Ab cocktail + Leica ChromoPlex™ 1 Dual Detection

K

L

CD3 – CD20 – Bcl 2 Leica Bond RTU + Leica Bond Refine Detection

CD20 Bcl2

CD3

1. Define the scope for validation

IHC + confirmation testing ISH - SISH

HER-2 (4B5) Roche RTU Confirm HER-2 + ultraView Universal DAB detection

HER-2 – Silver In situ Hybridization Roche Dual ISH HER-2 + SISH DNP Silver & Red DIG detection

1. Define the scope for validation

PCR IHC + confirmation testing

MSH 6 (clone 44) Becton Dickinson 1/400 + Leica Bond Refine Detection

2. Describe process for validation

▪ Process description : ▪ Main process ▪ Sub process ▪ Combination

▪ Method : ▪ Text ▪ Scheme : Word™, Visio™,…

= systematic overview process within scope

2. Describe process for validation

Sample

Histochemical Immuno Molecular

Histology Cytology

Procesflow !

2. Describe process for validation

Procesflow !

Request

LIS

Select

samples

Print

tasklist

LIS

Print

label

barcode

Sample on

coated slide

Controlslide

and/or

sample

Perform

Run

YES

NO

Store slide

<24 – 48u :RT

> 48u : -20°C

Load slides Load reagents

Start run

Scan

slides and

reagents

Perform

staining

Bluing &

mountingEnd of staining

Correct

reagents

Pathologist Reporting

Remove

reagents

GrossingSample

receptionSample

Microscopy

Processing &

embeddingSectioning

3. Define risk criteria

▪ General requirements : – Legal

– Standards : e.g. ISO 15189

▪ Practical experience, GLP

▪ Guidelines : e.g. ASCO/CAP, CLSI, …

▪ Literature

▪ Recommendations : – EQC organisations e.g. NORDIQC

– manufacturers

• Manufacturer

• Acceptance testing

• Optimalization

• Validation

• Maintenance

• Reagent management

• Training

Men Machine

Means Method

Input ? Depends on scope !

3. Define risk criteria

Available info

▪ ASCO/CAP HER-2 guidelines : – Fixation time 6-72 h

Used info

▪ Fixation time 6-72h

3. Define risk criteria : possible risks

▪ Determine possible sources of errors - risks ▪ What can go wrong ?

▪ Not complying to guidelines ?

▪ Risk identification : input ? ▪ Brainstorming

▪ Fault tree analysis

▪ Fishbone diagram

▪ …

3. Define risk criteria : possible risks

Fish bone diagram (Ishikawa)

Scope

Men

Method

Machine

Means

Primary

Secundary

Scope : cause & effect • Brainstorm • using fishboneand “M”

model 1st line risk 2nd line related risk

4. Perform risk assessment : FMEA

▪ FMEA : Failure Mode and Effects Analysis

▪Risk = chance x effect values ? scoring ?

Processtep Failure Consequence

Describe processtep What can go wrong ? What are the consequences ?

Grossing : • Time to fixation

Time to fixation > 1h

Impact on HER-2 staining

Processflow Fishbone diagram INPUT

▪ likelihood

▪ Probability

▪ Frequency

Common Terms Rating Example (ISO 14971)

Practical Example

Frequent 5 ≥ 1/1,000 > 1x/week

Probable 4 < 1/1,000 and ≥1/10,000

Once every few months

Occasional 3 < 1/10,000 and ≥1/100,000

Once a year

Remote 2 < 1/100,000 and ≥1/1,000,000

Once every few years

Improbable 1 < 1/1,000,000 and ≥10,000,000

Unlikely to ever happen

4. Perform risk assessment : scoring criteria

Rating : • 1-5 • 1-6 • 1-10 • …

▪ extent

▪ severity

Common Terms Rating Possible Description (ISO 14971)

Catastrophic 5 Results in patient death

Critical 4 Results in permanent injury of life-threatening injury

Serious 3 Results in injury or impairment requiring professional medical intervention

Minor 2 Results in temporary injury or impairment not requiring professional medical intervention

Negligible 1 Inconvenience or temporary discomfort

4. Perform risk assessment : scoring criteria

Rating : • 1-5 • 1-6 • 1-10 • …

Risk = chance x effect

+ Detectability

= Risk Priority Number

Priority & acceptance levels

Common Terms

Rating Example

Low 5 Control is ineffective

4 Control less likely to detect the failure

3 Control may or may not detect the failure

2 Control almost always detects the failure

High 1 Control can detect the failure

4. Perform risk assessment : scoring criteria

Patient safety

• Sample mismatch

• Staining mismatch

• Interpretation of results

Integrity of data

• Case, task : number

• Data transfer

Continuum of services

• Maintenance

• Stock management

• …

4. Perform risk assessment : scoring criteria

▪ Proces description (processflow)

▪ Risk criteria – input - scope (fishbone diagram)

▪ Risk / failure : patient safety, data integrity, continuity of services

Processtep Failure Consequence Chance Effect Risk Detection RPN

Describe processtep

What can go wrong ? What are the

consequences ? (1 – 6) (1 – 6)

(1 – 6)

Grossing : • Time to

fixation

Time to fixation > 1h

Impact on HER-

2 staining 2 6 12 2 24

4. Perform risk assessment : FMEA scoring

5. Select validation criteria

RPN (Risk Priority Number) : (= Risk x detection)

• Allows ranking

• Allows selection of validation items

• Level of detection possibilities : • Visual, manual

• Automated

5. Select validation criteria

RPN (Risk Priority Number)

Unacceptable 60 Class 1

High 21 - 60 Class 2

Medium 11 - 20 Class 3

Low 1 - 10 Class 4

RPN Class Further action required ? Acceptance ?

Class 1 Direct action No

Class 2 Short term action No

Class 3 Middle to lang term action Yes, within context Klasse 4 None Ja

Validation criteria !

Determine acceptance level !

6. Perform validation

▪ FMEA : Selected risks - RPN = validation

▪ Use available information, including : – Validation of instruments

– Validation of methods

– Keeping instrments & methods in a validated state

▪ Perform validation on “new” items

▪ Determine acceptance criteria validation

7. Maintain in a validated state = active !

▪ Instruments : – Validation – Maintenance – Control T°

▪ Methods : – Validation – Controls

▪ Proces validation monitoring

▪ Change management

Content

▪ Background

▪ Definitions

▪ Process validation model

▪ Practical example

Scope = HER-2 testing

Request

LIS

Select

samples

Print

tasklist

LIS

Print

label

barcode

Sample on

coated slide

Controlslide

and/or

sample

Perform

Run

YES

NO

Store slide

<24 – 48u :RT

> 48u : -20°C

Load slides Load reagents

Start run

Scan

slides and

reagents

Perform

staining

Bluing &

mountingEnd of staining

Correct

reagents

Pathologist Reporting

Remove

reagents

GrossingSample

receptionSample

Microscopy

Processing &

embeddingSectioning

▪ Input - plug in :

▪ Guidelines : Belgian guidelines on HER-2 testing, ASCO/CAP

▪ Literature references

▪ Guidelines from manufacturer

▪ Good laboratory practice

▪ Validation of instruments & methods

HER-2 testing : risk criteria

HER-2 testing : risk criteria

HER-2 testing : FMEA risk assessment

▪ FMEA :

Code Process step Failure Consequence Chance Effect Risk grade Detection RPN

Score Number % Follow up ?

Unacceptable 1 1,67% Yes

High 13 21,67% Yes

Medium 8 13,33% No

Low 38 63,33% No

Total 60 100%

HER-2 testing : FMEA : results

▪ FMEA : 14 validation points : – 1 unacceptable

– 13 high risk

▪ = validation items

▪ Risk reducing measures incorporated in validation

HER-2 testing : validation criteria

▪ Validation of instruments

▪ Validation of methods

▪ Validation of LIMS

▪ Internal quality control

▪ Acceptance testing critical reagents

▪ Monitoring critical temperatures : – Stretching tables

– Paraffin stations

– Refrigirator & freezer

Allready validated !

▪ Maintaining the validated state of instruments

▪ Maintaining validated state of methods

▪ Appropriate use of appropriate controls

▪ Monitoring critical process point

HER-2 testing : Maintaining a validated state

▪ Process validation will - in a systematic and documented way - highlight critical points within the scope of the IHC process. The validation and follow up of these points will, toghether with the validated IHC method, be an aid to the standardization of the total IHC process.

Conclusion

donald.vanhecke@stlucas.be

THANK YOU !