Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

IL8 and EDEM3 gene expression ratio indicates peripheral blood mononuclear cell (PBMC) quality.

Author(s): Kofanova O, Bellora C, Aguilar-Quesada R, Bulla A, Panadero-Fajardo S, Keipes M, Shea K, Stone M, Lescuyer P, Betsou F

Publication: J Immunol Methods, 2019, Vol. 465, Page 13-19

PubMed ID: 30496732 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the effects of the duration and temperature of pre-centrifugation storage of blood collected in tubes containing different anticoagulants (K2EDTA, Na citrate, or Li heparin) on the integrity of RNA isolated from peripheral blood mononuclear cells (PBMCs). Blood was collected from seven different cohorts that included a total of 50 healthy volunteers, 18 patients diagnosed with rheumatoid arthritis, 10 patients diagnosed with HIV, and four patients being treated for acute pancreatitis.  Whole blood was collected in tubes containing K2EDTA, citrate (CPT or ACD tubes), and lithium heparin tubes that were subjected to a pre-centrifugation delay either at room temperature or 4°C for 30 min, 2-4 h, 3 h, 23-24 h, or 48 h.  PBMCs were separated from blood by Ficoll and centrifugation at either 1800 or 2000 g for 20 min. PBMC aliquots were stored at -80°C until RNA extraction with the miRNeasy Mini kit. RNA concentration was determined by spectrophotometry, integrity was determined by the bioanalyzer-generated RNA Integrity Score (RIS), and transcript levels were quantified by the TaqMan Gene Expression Assay following reverse transcription with random primers for interleukin 8 (IL-8), endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 3 (EDEM3), and RNA polymerase II (POLR2A).  The “PBMC preanalytical score” developed by the authors was calculated based on the ratio of -∆∆Ct values of each transcript of interest (IL-8, EDEM3) relative to the normalizer (POLR2A).

   Summary of Findings:

   The range of RNA integrity scores (RIS) observed among specimens collected in each anticoagulant overlapped regardless of the temperature of the pre-centrifugation delay (EDTA at 4°C=7.2-8.2, EDTA at RT= 6.8-8.1; CPT at 4°C=7.1-8.5, CPT at RT =6.4-8.5).  While the authors state that lower RIS were observed with longer pre-centrifugation delays, data was not provided.

   The diagnostic performance of the “PBMC preanalytical score” developed by the authors, which is based on the relative ratios of normalized levels of IL-8 and EDEM3 to POLR2A, was determined for each anticoagulant.  For blood collected from healthy individuals into EDTA tubes, the sensitivity and specificity of the PBMC preanalytical score discriminating between a pre-centrifugation delay of ≤3 h and ≥24 h was 99% and 98%, respectively, when the delay occurred at 4°C, and 97% and 98%, respectively, when the delay occurred at room temperature.  Both the sensitivity and specificity of the PBMC preanalytical score were 100% among EDTA blood collected from patients diagnosed with HIV or an inflammatory illness, although the discernable delay was extended to ≥48 h and the cut-off value was substantially higher than for healthy individuals (67.6 and 1258 vs 28, respectively). For blood collected from healthy individuals into CPT tubes, the sensitivity and specificity for discriminating samples that experienced a pre-centrifugation delay ≥24 h at room temperature using the PBMC preanalytical score was 80% and 84%, respectively, but increased to 90% and 91%, respectively, when the delay was shifted to ≥48 h.  When CPT tubes were used, the sensitivity and specificity of the PBMC preanalytical score for discerning a pre-centrifugation delay ≥48 h were both 100% when blood was sampled from patients diagnosed with HIV, but was 78% and 95%, respectively, when sampled from patients diagnosed with an inflammatory condition. Similar to EDTA blood specimens, citrate anticoagulants necessitated higher PBMC preanalytical score cut-off values for patients diagnosed with HIV and inflammatory conditions compared to healthy patients.  A similar concomitant increase in PBMC preanalytical score and pre-centrifugation delay was also observed in blood collected into tubes containing heparin (score= 6-18 at 3 h, 341-615 at 24 h, 2854-3462 at 48 h).

   Biospecimens

   • Cell - White Cells

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Other diagnoses
   • AIDS/HIV-related
   • Rheumatoid Arthritis

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	Potassium EDTA Sodium citrate Lithium heparin
   Biospecimen Acquisition	Type of collection container/solution	K2EDTA Na citrate Li heparin
   Real-time qRT-PCR Specific	Targeted nucleic acid	IL-8 EDEM3
   Storage	Storage temperature	4°C Room temperature
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Storage duration	30 min 2-4 h 3 h 23-24 h 48 h

   View more