The Imprecision in Precision Medicine

Right now, precision medicine is beset with an insidious imprecision: namely, the control of the preanalytical steps that impact the quality and integrity of patient biospecimens from which all molecular data is derived. In short, there is a garbage-in, garbage-out problem that has not yet been adequately addressed. 

Clinical specimens are collected, handled, and processed according to routine procedures in the practice of medicine, surgery, interventional radiology, and pathology. All procedures, practices, and environmental factors to which the biospecimen is subjected prior to a laboratory analysis are known as preanalytical factors. Artefactual alterations caused by these factors may skew the data from molecular analyses, render the analysis data uninterpretable, or even preclude analysis altogether if the quality of specimen is severely compromised. As a result, patient care and safety may be affected¹, and medical research dependent on patient samples may be compromised.

Despite these issues, there is no requirement to control or document preanalytical variables in routine clinical practice in pathology with the single exception of breast cancer tissue that is to be assayed for estrogen/progesterone receptor expression and/or HER2 protein over-expression. In that single context, the preanalytical steps of cold ischemia time, formalin fixation method, and total time in formalin are addressed in the guideline jointly developed by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) and are included in the Accreditation Checklist of the CAP Laboratory Accreditation Program (LAP). 

Several national and international standards organizations have published guidelines for the preanalytical steps that are recognized to be critical for valid, reliable molecular testing (see the Top Six Lists), but compliance with any of these guidelines is voluntary unless they are part of an accreditation program such as ISO 15189 Medical Laboratory Accreditation. As a result, existing guidelines for biospecimen acquisition, handling and processing are inconsistently applied, either in clinical practice or research settings. Compliance is further reduced because most authoritative guidelines are proprietary and are not freely available to all members of the biomedical community.

Recognizing the widespread and growing importance of molecular data derived from patient specimens, especially patients with cancer, the Personalized Healthcare Committee (PHC) established the Preanalytics for Precision Medicine Project Team (PPMPT) to develop a basic set of recommendations from the literature for preanalytics for both tissue and blood specimens that could be implemented in routine pathology practice.  In a four-year effort, the PPMPT reviewed all relevant scientific data on the impact of preanalytical steps on nucleic acids and proteins in human tissue and blood specimens. This work informed a set of recommendations for key preanalytical factors for tissue and for blood-based on current literature . If these recommendations are widely employed, the preanalytical factors having the strongest detrimental effect on the molecular integrity of patient biospecimens would be both controlled and documented, and the fitness for molecular analysis of patient specimens would be assured. 

Currently, biospecimens from cancer patients present the most critical need for preanalytical quality control because they so frequently undergo molecular testing. Therefore, the PPMPT recommends a stepwise approach to addressing preanalytical control and documentation, beginning with all specimens from cancer patients. As molecular testing becomes more widely employed, these same practices will need to be employed more broadly. 

The PPMPT verified that a small set of preanalytical factors cause most of the molecular compromise in tissue and blood specimens. Controlling these would not optimize specimens for any particular type of molecular analysis but would create a known baseline level of molecular quality control where none now exists. These recommendations are consistent with comparable recommendations from other authoritative sources that have issued voluntary guidelines based on published based data. The last, but certainly not least, of the recommendations is documentation of the key preanalytical steps to provide a record of the specimen provenance that will be part of the pathology report. Even when a practice recommendation cannot be met, deviations from the recommendations can, at the very least, be recorded.

The Top Six lists of practice recommendations for control and documentation of the important preanalytical variables are summarized below.

1. Time to Stabilization (cold ischemia time)

• One hour or less

2. Method of Stabilization

• Fixative: 10% phosphate-buffered formalin, pH 7.0
• Total time in formalin: at least 6 hours, not more than 24-36 hours (tissue with high-fat content may require 48 hours) 
• Acid decalcification, before or during stabilization, is contraindicated for nucleic acid analyses

3. Method of Processing 

• Specimen thickness not to exceed 4-5 mm 
• Volume:mass ratio 4:1 at a minimum, preferably 10:1, with tissue completely submerged 

4. Tissue Processor Variables

• Processor maintenance daily per manufacturer’s recommendations 
• Quality of processing fluids rigorously maintained 
  • Maintenance of formalin purity and pH
  • Attention to water (i.e., formalin) contamination of alcohol baths 
• Type of paraffin
  • Low-melt paraffin (melts at <60°C)

5. Storage Conditions

• Dry, pest-free conditions at room temperature (defined as 18-25°C)

6. Documentation Data 

• Documentation data for the above factors and/or deviations from the recommendations

Note: Tissues specimens considered unacceptable for molecular testing include desiccated tissues or those known to have been improperly collected or stored.

1. Time to First Processing Step

• <60 minutes (unless EDTA or specialty cell-stabilization tube used)
  • 4-6 hours for EDTA tube
  • ≤48 hours for cell stabilization tube

2. Method of Acquisition

• Tube type: (specialized for a specific molecule species vs. not)
  • If processing time is >2-3 hours, use acid-citrate-dextrose (ACD) tube
  • Ethylenediaminetetraacetic acid (EDTA) tube for nucleic acid studies, proteomics studies or circulating cell-free nucleic acid studies (cell-free nucleic acid analysis requires rapid stabilization or potentially specialty cell-stabilization tubes)

• Volume of tube fill 
  • Complete fill per manufacturer’s recommendation
  • Tube inversions per manufacturer’s recommendations (typically 10)

• Draw order
  • Culture tube
  • Non-additive tube
  • Coagulation tube (sodium citrate)
  • Clot activator tube
  • Clot activator and serum separator tube (clot activator plus separator gel)
  • Heparin tube (lithium or sodium heparin)
  • EDTA (ethylenediaminetetraacetic acid) tube
  • Tubes with other additives (e.g., sodium fluoride; acid-citrate-dextrose; other)

3. Method of Stabilization

• Tube inversions per manufacturer’s recommendations 

4. Method of Processing

• Centrifugation speed/time per validated protocol and biomolecule being studied
• Temperature: Room temperature (defined as 18-25°C) unless validated protocol dictates otherwise 

5. Storage Conditions

• ≤1 freeze-thaw cycle: for nucleic acids and proteins use aliquots

6. Documentation Data

• Documentation data for the above factors and/or deviations from the recommendations

Note: Blood specimens considered unacceptable for molecular testing include hemolyzed samples or those known to have been improperly collected or stored.

Definitively addressing the preanalytics challenge has been a long time in coming. It may not be easy in all practice settings, but there is little doubt that it is the right thing to do. As we move forward towards precision medicine, greater preanalytical precision will be an absolute necessity. The PPMPT would argue that it is a necessity right now, and urgently requires our commitment to ensuring its implementation. The CAP is taking a giant step towards greater precision in precision medicine by submitting the PPMPT recommendations to the CAP Laboratory Accreditation Program for possible inclusion in the future. 

¹ Compton CC, Robb JA, Anderson MW, et al.  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.