Preanalytics: The Imprecision in Precision Medicine...and biologically reactive Molecular composition subject to further alteration/degradation A N A L Y S I S S T O R A G E ... Cold

© College of American Pathologists

Carolyn Compton, MD, PhD, FCAP August 13, 2020

Preanalytics:The Imprecision in Precision MedicineThe Challenge Before Us;The Solutions Ahead


Webinar Host

• This series is sponsored by the Personalized Healthcare Committee (PHC)

• Today’s webinar host is Frederick Eyerer, MD

13 August 2020 2


Housekeeping

• This presentation will be recorded. The recording and PDF will go out to all registrants in one week

• All lines are muted during the presentation

• Please send in your questions as you think of them via the “Question Box” in your control panel

13 August 2020 3


Carolyn Compton, MD, PhD, FCAP• Academic pathologist specializing in

gastrointestinal disease.

• Professor of Life Sciences at Arizona State University and Professor of Laboratory Medicine and Pathology at the Mayo Clinic School of Medicine.

• Adjunct Professor of Pathology at the Johns Hopkins Medical Institutions.

• Research interests include biospecimen science and the development of prognostication tools for cancer patients.

13 August 2020 4


Disclaimer

• The CAP does not permit reproduction of any substantial portion of the material in this Webinar without its written authorization. The CAP hereby authorizes attendees of the CAP Webinar to use the PDF presentation solely for educational purposes within their own institutions. The CAP prohibits use of the material in the Webinar – and any unauthorized use of the CAP’s name or logo – in connection with promotional efforts by marketers of laboratory equipment, reagents, materials, or services.

13 August 2020 5


Disclaimer, continued

• Opinions expressed by the speaker are the speaker’s own and do not necessarily reflect an endorsement by the CAP of any organizations, equipment, reagents, materials, or services used by participating laboratories.

13 August 2020 6

© College of American Pathologists 7

The Challenge Before Us;The Solutions Ahead

• Molecular analysis of patient specimens: the foundation of precision medicine

• The critical role of preanalytics in molecular testing

• The state of the science and the experience of experts

• Defining a solution

• Next steps and a vision for the future

13 August 2020


Unianalyte Tests

Multianalyte Tests

Networks and Systems

Evolution Of Biomarker Testing In The “Omics Era”

Omics Analyses

Technology Development Has Driven the Evolution of Molecular Analysis

© College of American Pathologists 13 August 2020 9

The Complexity of Laboratory Analysis Is Destined to Increase Further

• Complex molecular biomarkers raise the bar for biospecimen quality and consistency.

• Going forward, the requirement to generate data necessary to understand higher levels of biological function will increase.

• Their specimens are the patients’ investment in the future, as molecular analysis may be needed on archived specimens.

• Currently, exceedingly few biospecimens meet these challenges!


• It is impossible to build precision medicine on a foundation of widespread imprecision related to patient specimens.

• High-quality test results depend on high-quality test analytes from biospecimens as starting materials.

• The control of specimen quality involves every step in the supply chain and every person in the chain of custody.

The Vision of Precision Medicine

1013 August 2020








13 August 2020


• Definition: all factors and steps that precede a specimen analysis

• May artefactually alter analytes; altered analytes alter test results

• May cause two different types of molecular alterationo Molecular content (via stress responses in viable specimens)

o Molecular integrity

• Requires rigorous real-time, up-front management o You can’t go back and restore lost quality

What Is/Are “Preanalytics”

13 August 2020 12


Patient Acquisition Handling/Processing Storage Distribution Scientific

AnalysisMedical/SurgicalProcedures

RestockingUnusedSample

Pre-analytical Factors Affect BothMolecular Composition and Molecular Quality

Time 0

Post-acquisitionPre-acquisition

Factors (examples):� Time at room temperature� Temperature of room� Type of fixative� Time in fixative� Rate of freezing� Size of aliquots

Factors (examples):� Antibiotics� Other drugs� Type of anesthesia� Duration of anesthesia� Arterial clamp time

Specimen is viableand biologically reactive

Molecular composition subject to further alteration/degradation

ANALYS I S

STORAGE

13 August 2020 13


HER2/CEP17 = 0.98

HER2/CEP17 = 0.29

Do Preanalytics Matter? Everyone Agrees It Does.

Common laboratory artifacts caused by inappropriate sample collection and transport: how to get the most out of a sample. Gilor and Gilor. Top Companion Anim Med. 2011 May;26(2):109-18. doi: 10.1053/j.tcam.2011.02.003.

Preanalytical challenges – time for solutions. https://doi.org/10.1515/cclm-2018-1334 Received December 17, 2018; accepted January 8, 2019

Pre-analytical issues in the haemostasis laboratory: guidance for the clinical laboratories. Magnette et al. Thrombosis Journal 2016

The cost of poor blood specimen quality and errors in preanalytical processes. Sol F. Green. Clinical Biochem 2013. Stony Brook School of Medicine

Where Are the Preanalytical Stability Standards?Corinne R. Fantz, Dina N. Greene DOI: 10.1373/jalm.2018.026062 Published April 2018

13 August 2020 14


HER2/CEP17 = 0.98

HER2/CEP17 = 0.29

Indirect Evidence

ORIGINAL ARTICLES Variable Specimen Handling Affects Hormone Receptor Test Results in Women With Breast Cancer: A Large Multihospital Retrospective StudyFlory L. Nkoy, MD, et al. Arch Pathol Lab Med 2010; 134: 606-612.

Frequency of ER/PR negativity was calculated for each surgery day and compared among hospitals and between 2 groups: • Regular (specimens obtained Sunday through Thursday, more likely to be

tested within 24 hours of surgery) • Prolonged (specimens obtained on Friday and Saturday, more likely to be

tested more than 24 hours after surgery) specimen handling conditions.

Estrogen receptor and PR negativity remained highly variable among hospitals despite use of a single laboratory and tended to be significantly associated with prolonged specimen handling.

Intermountain Healthcare, Utah

Conclusion for patients: Don’t have your breast surgery on Friday!

13 August 2020 15


Current State of Patient Specimen Quality in the USA: ~ 30% Are High Quality

13 August 2020 16


Powerful Technologies Bring Powerful Risks

13 August 2020 17

• No technology can spin straw into gold – you must begin with gold!

• The technological capacity exists to produce low-quality data from low-quality analytes with unprecedented efficiency.

• We now have the ability to get the wrong answers with unprecedented speed.

© College of American PathologistsKhoury T, et al., Mod Pathol. 2009 Nov;22(11):1457-67

a

c

HER2/CEP17 = 0.98

HER2/CEP17 = 0.29

30 min

2 hr

4 hr delay

HER2 IHC and FISH in Breast Cancer:Loss of Signal with Time to Fixation

pMAPK IHC of Colon Cancer : Gain of Signal with Time to Fixation

10 min

20 min

60 min

Hartmut Juhl, Indivumed GmbH, BRN

Cold Ischemia Time Affects Molecular Assay Results

13 August 2020 18


Gene ExpressionPre vs. Post Surgery

(Number of Gene Changing Expression)

Protein ExpressionPre vs. Post Surgery

(% Change)

David DA, et al. Oncotarget 2014 Nov 30;5(22):11017-28. doi: 10.18632/oncotarget.26

The variability of gene expression was significantly higher in cancer tissue than normal tissue.

>2-fold Change in Expression of 15% of Genes and 60% of Proteins with Warm Ischemia Time

13 August 2020 19


ProcessingProcedure,Temperatureand Time

Blood Draw Procedure

CollectionTubes and Order ofdraw

Distribution& Storage

MolecularAnalysis

Patient Consent and Preparation

Blood Collection and Plasma Processing: Biomarkers and Circulating Tumor Cells

13 August 2020 20

http://images.google.com/imgres?imgurl=http://www.rsc.org/images/microarrays-200_tcm18-44617.jpg&imgrefurl=http://www.rsc.org/chemistryworld/restricted/2006/January/Microarrays.asp&h=154&w=200&sz=52&hl=en&start=6&um=1&tbnid=j26Wb2jpsiLAyM:&tbnh=80&tbnw=104&prev=/images?q=microarrays&svnum=10&um=1&hl=en&rls=SUNA,SUNA:2006-49,SUNA:en&sa=N


Preanalytical Variables and Blood/Plasma

� Tourniquet vs. none

� Tourniquet time

� Central line vs. peripheral vein

� Draw order

� Tube type

� Tube volume

� Tube inversions

� Total time of draw

� Vacuum, butterfly vs. syringe

� Needle bore

� Type of port (if used for access)

� Tube agitation during transport*

� Time to centrifugation

� Centrifuge speed

� Centrifuge temperature

� Number of centrifugations

� Use or not of discard tube

� Temperature, duration storage 13 August 2020 21


Biospecimen Quality Drives Molecular Diagnosis

Biospecimen Analysis Biospecimen Collection

Biospecimen processing and ba

Molecular Data Diagnosis / TherapyDiagnostics / Therapeutics Development

PRECISION MEDICINE

Biospecimen Handling and Processing

QUALITY HERE

Starts with the Patient

Ends with the Patient

DETERMINES QUALITY HERE

13 August 2020 22


2000 2010 2020

Sample collection and collection design

Sequencing

Data reductionData management

Downstream analysis

Are pathologists prepared for what’s coming with the changing focus on samples?

From Ken Bloom, MD, GE Healthcare, June 2014

Estimating the Changing Aspects of NGS

13 August 2020 23


• Place where test is doneo CLIA/CAP laboratory accreditation

• People doing the testo Educationo Proficiency testingo Licensure

• Platforms used for testingo CDRH approved devices

• Processes followed for testingo SOPs o Quality management

• Patient samples to be testedo One enforced standard: ASCO/CAP guideline breast cancer

Rigor and Reproducibility in the Pathology Lab: How Is It Assured?

13 August 2020 24


Rigor and Reproducibility for Biomarker Measurement in the Clinical Lab: How Is It Assured?

More is known about the quality of beef in

supermarkets than about the quality of human biospecimens used in molecular testing

because the USDA regulates the quality of beef.

13 August 2020 25








13 August 2020


Biomarker Tests for Molecularly Targeted Therapies: Key to Unlocking Precision Medicine

National Academies Press, Jun 30, 2016

• One of the 10 stated goals is to specifically address biospecimen quality.

• Goal 9) Enhance specimen handling and documentation to ensure patient safety and the accuracy of biomarker test results.

“The reliability of biomarker test results depends on the quality of the patient specimens.”

“Professional organizations and health care institutions should develop and implement standards for obtaining adequate specimens.”

The National Academy of Sciences, Engineering and Medicine (IOM) Speaks

13 August 2020 27


Biospecimen

13 August 2020 28


• Academic genomics experts (scientists: basic and translational)• Academic proteomics experts (scientists: basic and translational)• Expert molecular pathologists• CAP leadership:

o Presidento President Electo Immediate Past President

• Surgeons• Patient advocacy groups• Funders: NCI, advocacy organizations• Regulators: FDA• Leadership of professional societies: ASCO, AACR• Payers: CMS, Palmetto, Aetna, BC/BS• Industry (Pharma, Platform manufacturers, Tissue providers):

Illumina, Genetech, Caprion, Indivumed, Becton-Dickenson, Novartis, Abbott

55 Experts Representing All Stakeholder Groups and Points of View

13 August 2020 29


Goal:

• Agree on the pre-analytical steps in the biospecimen lifecycle that MOST compromise the quality of tissue and blood

– “Top 10 List”

• Identify where the greatest value can be delivered in the control of pre-analytical variation (biggest quality bang for the buck)

• “Top 5 List” + Metadata

NBDA Convergence Conference 2014

13 August 2020 30


1. Time to stabilization

2. Method of stabilization

3. Method of processing

4. Tissue processor variables

5. Storage conditions

6. Documentation of all above

1. Time to processing

2. Method of acquisition



5. Storage conditions

6. Documentation of all above

Tissue Blood/Plasma

The CAP PPMPT Verifies the Consensus Lists

13 August 2020 31








13 August 2020


• CAP Preanalytics for Precision Medicine Project Team (PPMPT)• Verification of the Top 5 lists - review human literature and

benchmark comparisons with CLSI, ISBER, NCI, ISO• Establish professional performance metrics for Top 5

o Data-driveno Practical

• Educate pathology workforce and professionals upstream of pathology in the quality chaino Preanalytics Summit Conference, December 2019

• Implement and enforce performance metrics through the CAP LAP• COC enforcement outside of pathology • Seek reinforcement through FDA guidance, research funder

requirements, pharma companies with companion diagnosticso Personalized Medicine Coalition working group formed

Actions

13 August 2020 33


Compton CC, Robb JA, Anderson MW, Berry AB, Birdsong GG, Bloom KJ, Branton PA, Crothers JW, Cushman-Vokoun AM, Hicks DG, Khoury JD, Laser J, Marshall CB, Misialek MJ, Natale KE, Nowak JA, Olson D, Pfeifer JD, Schade A, Vance GH, Walk EE, Yohe SL. Preanalytics and Precision Pathology: Pathology Practices to Ensure Molecular Integrity of Cancer Patient Biospecimens for Precision Medicine. Arch Pathol Lab Med. 2019; 143:1346-1363.

“Fast” Forward Five Years

PPMPT now revising all CAP LAP

checklist

13 August 2020 34

https://www.ncbi.nlm.nih.gov/pubmed/31329478


1. Time to stabilization: 60 minutes or less


o Section thickness: ≤5 mm

o Volume formalin/size tissue ratio: ≥4:1 (optimal ≥10:1)


o Type of fixative: 10% neutral phosphate-buffered formalin

o Time in fixative: 6-36 hours (optimal 24) - greater for fatty tissues

4. Tissue processor variables

o Maintenance schedule: Manufacturer’s recommendation

o Paraffin type: low melt <60°C

o Forbid non-standard practices (“topping off with non-standard solutions)

5. Storage conditions: Room temperature (e.g., 20-25°C); dry; pest-free

6. [Metadata to be collected]: Any deviation from the above recommendations

PPMPT Verified Recommendations: Tissue

13 August 2020 35


1.Time to first processing step: ≥ 60 min or less (4-6 hr EDTA tube / ≥48 hr cell stabilization tube)

2.Method of acquisitiono Tube type appropriate for test

o Volume / tube fill / mixing: complete fill; 10 inversions additive mixing

o Draw order: order specified per WHO 2010 (contamination precluded)

3.Method of processingo Centrifuge time/speed: validated protocol per molecule being measured

o Temperature: room temperature (e.g., 20-25°C)

4.Storage conditions: 1 freeze-thaw only (nucleic acids/proteins use aliquots)

5.Documentation data: for above and any deviation from the above Compton et al. Preanalytics and Precision Pathology; Pathology Practices to Ensure Molecular Integrity of Cancer Patient Biospecimens for Precision Medicine. Archives Pathol Lab Med July 2019; doi: 10.5858/arpa.2019-0009-SA.

PPMPT Verified Recommendation: Blood

13 August 2020 36







• Next steps and going forward

13 August 2020


Garbage in…

…Garbage out

The Challenge

13 August 2020 38


It’s Not Just the Pathologists

Links in the quality chain:

• Surgeons

• Nurses

• Transport personnel

• Phlebotomists

• Interventional radiologists

13 August 2020 39


Call to Action and Desired Future State

• Call to action for all professionals in the specimen quality

chain• Seamless chain of custody from patient to analysis

• Enforcement through Joint Commission and/or COC

• Billing codes from payers• Grants requirements from NIH and advocacy

• FDA guidance

• Transformative impact on the quality of molecular analyses for patient care and translation research

• Reduction of irreproducibility in medicine and science

13 August 2020 40


It Won’t Be the First Time Pathologists Lead the Way

13 August 2020 41


Medicine, Science and The Right Thing to Do

It’s for our patients.

13 August 2020 42


Thank You

• Fellow CAP members and pathology colleagueso Members of the CAP Personalized Healthcare Committee

from 2015 – presento Members of the Preanalytics for Precision Medicine Project

Team from 2015 – presento CAP Presidents 2010 – present

• CAP Staff:o PHC 2015 – presento Librarianso CAP LAP

• Everyone listening today

13 August 2020 43


CAP’s Precision Medicine Webpage

• The webpage includes brief, relevant articles by CAP members that enable the reader to gain a better understanding of a particular area of precision medicine.o Examples include pharmacogenetics, immune response genes, and the latest

in the molecular drivers of cancer.

o Access them www.cap.org > Member Resources > Precision Medicine

13 August 2020 44

http://www.cap.org/


• The CAP has created the Pathology Resource Guides to assist pathologists in understanding key emerging technologies. o Printed guides are now available for members ($39) and non-members ($69)o The digital copy of the Resource Guides are a complimentary member benefito Access them www.cap.org > Resources and Publications

CAP’s Pathology Resource Guide: Precision Medicine

© 2018 College of American Pathologists. All rights reserved.

13 August 2020 45

http://www.cap.org/


THANK YOU!

Thank you for attending our webinar,“Preanalytics: The Imprecision in Precision Medicine”

by Carolyn Compton, MD, PhD, FCAP

For comments about this webinar or suggestions for upcoming webinars, please contact

[email protected].

NOTE: There is no CME/CE credit available for today’s free webinar. The PDF of the presentation will be sent out in a week.

13 August 2020 46

mailto:[email protected]


Preanalytics: The Imprecision in Precision Medicine...and biologically reactive Molecular composition subject to further alteration/degradation A N A L Y S I S S T O R A G E ... Cold

Documents