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External Quality Assurance programs for processing methods provide evidence on impact of preanalytical variables.

Author(s): Verderio P, Ciniselli CM, Gaignaux A, Pastori M, Saracino S, Kofanova O, Betsou F

Publication: N Biotechnol, 2022, Vol. 72, Page 29-37

PubMed ID: 36049650 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the potential effects of preanalytical factors on the analysis of DNA, RNA, cfDNA and PBMC from blood; DNA and RNA from FFPE tonsil specimens; and DNA from saliva and stool by comparing data obtained at several facilities, each of which used their own workflow The preanalytical factors examined included storage temperature, extraction kit/automation, elution buffer, inclusion of a DNAse/RNAse step, centrifugation parameters and PBMC freezing parameters. This study included data obtained over a 6 year period from 997 participants/representing different processing methods. For each analyte, a PAXgene tube of whole blood (patient characteristics were not specified) was distributed to each participating institution (one tube/analyte): DNA (202-303 participants), RNA (102-127 participants) and cfDNA (34-38 participants). In addition, each participant (74-111 for DNA and 59-62 for RNA) received four 10 µm thick sections of FFPE tonsil (two for DNA and two for RNA), an Omnigene Oral tube of saliva from a healthy volunteer (19-23 participants), and an Omnigene Gut tube containing stool from a healthy volunteer (26-34 participants). DNA and RNA were extracted by each participant by their own methods. Each participant (139-143) collected blood from a healthy volunteer using their anticoagulant of choice for isolation of PBMCs using the method of their choice. DNA was evaluated by spectrophotometry, fluorometry, TapeStation, and long-range and multiplex PCR (details not specified). RNA was evaluated by spectrophotometry, and bioanalyzer. PBMCs were evaluated by flow cytometer, ELISpot, and cell counter.

   Summary of Findings:

   In blood specimens, DNA extraction method had a significant effect on total DNA yield (P=0.003), with higher yields observed when extraction was with a magnetic bead-based method rather than silica-membrane, salting out, or the “other’ method. Elution of DNA isolated from blood with Tris-based AE buffer led to higher yields than elution with water or TE (P=0.035). DIN of DNA from blood was significantly affected by extraction method (P<0.001) and extraction equipment (P=0.001), with the highest DIN observed in DNA isolated from blood using salting out methods (rather than silica membrane magnetic bead based or “other” methods) and the lowest when automation was employed. All but four DNA extracts from blood allowed for amplification of a long PCR product (length not specified) and of these the only identified commonality was lack of RNAse step. RNA purity was significantly affected by extraction method (P<0.0001) and inclusion of a DNAse step (P<0.0001), with higher purity observed when silica-column based methods were used versus magnetic-bead based or “other“ methods and when a DNAse step was included.

   DNA and RNA extracts from FFPE tissues had higher DNA purity and RNA quality indices when silica-based methods rather than magnetic-bead based methods were used for extraction (P<0.001 and P=0.005, respectively), higher DNA yields when a Tris-based elution buffer was used rather than water (P=0.005), and higher RNA yields when one FFPE tissue section was used rather than two (P=0.0001).  

   PBMC viability was highest when the first centrifugation was for ≤20 min rather than >20 min (significance not provided). No clear effect of anticoagulant (sodium heparin, K₂EDTA, Citrate dextrose, CPT, K₃EDTA or other), freezing method (Mr. Frosty, progressive rate freezer or other), braking during centrifugation, number of washing steps (1-2 versus 3-4), automated versus manual isolation, or centrifugation speed (≤500, 500-1000, or ≥1000 g) was observed on PBMC viability.

   Sample size limited the power of statistical analyses of stool and saliva specimens and cfDNA isolated from blood. However, DNA yields from saliva were higher when extraction was automated and the variability in saliva DNA yield was lower when saliva was stored at room temperature. There was no clear effect of elution buffer or extraction kit type on saliva DNA endpoints. DNA yields from stool were higher when DNA was extracted with a magnetic bead-based method rather than silica-column based kits but the authors state this is mostly attributable to a single outlier. The authors found no clear effects of any of the preanalytical factors (storage temperature, centrifugation temperature/speed/duration/braking, extraction method/kit., elution buffer, and use of RNAse) examined on cfDNA endpoints.

   Biospecimens

   • Cell - White Cells
   • Bodily Fluid - Blood
   • Bodily Fluid - Saliva
   • Bodily Fluid - Feces
   • Tissue - Tonsil

   Preservative Types

   • Formalin
   • PAXgene
   • Frozen

   Diagnoses:

   • Not specified
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Fluorometry
   DNA	Spectrophotometry
   DNA	PCR
   Cell count/volume	Immunoassay
   RNA	Fluorometry
   Cell count/volume	Light microscopy
   Cell count/volume	Flow cytometry
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	Plasma preparation tubes (PPT) Multiple forms evaluated Potassium EDTA Sodium heparin Citrate-phosphate-dextrose
   Biospecimen Aliquots and Components	Centrifugation	With braking Without braking Multiple speeds compared Multiple temperatures compared Multiple durations compared
   Biospecimen Preservation	Cooling or freezing method/ rate	Mr Frosty Multiple steps in controlled rate freezer
   Analyte Extraction and Purification	Analyte isolation method	Manual Automated Silica-membrane based method Salting out based method Magnetic-bead based method "Other" method
   Biospecimen Aliquots and Components	Aliquot size/volume	1 section 2 sections
   Analyte Extraction and Purification	Rehydration of dried sample/specimen	TE AE Water
   Analyte Extraction and Purification	Nucleic acid digestion	DNAse added RNAse added No DNAse No RNAse

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