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Banking of Tumor Tissues: Effect of Preanalytical Variables in the Phase of Pre- and Postacquisition on RNA Integrity.

Author(s): Zheng XH, Zhou T, Li XZ, Zhang PF, Jia WH

Publication: Biopreserv Biobank, 2022, Vol. , Page

PubMed ID: 35377214 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared RINs of RNA obtained from case-matched HCC and normal adjacent specimens subjected to up to 8 h of cold ischemia time at room temperature prior to preservation with RNAlater and freezing, case-matched HCC tumor specimens subjected to up to 8 freeze-thaw cycles, and HCC specimens stored at -80°C for 0-10 years. The authors also investigated the influence of patient age (<50 versus >50 years), gender, clinical stage, tumor location, HBV infection, tumor grade, tumor diameter, and surgical approach (open versus laparoscopic colectomy) on RIN. Case-matched tumor and normal adjacent tissue specimens were collected from 146 patients with hepatocellular carcinoma. Unless otherwise specified, specimens were divided and immediately (within 15-40 min of excision) placed in RNAlater overnight at 4°C after which RNAlater was removed and the specimen was frozen at -80°C until RNA extraction.  To evaluate the influence of patient age, gender, clinical stage, tumor location, HBV infection, tumor grade, tumor diameter, and surgical approach, specimens from 42 patients with HCC were used. To investigate the effects of cold ischemia, HCC and normal adjacent specimens from 8 patients were divided and subjected to a cold ischemia time of <30 min, 1, 2, 4, 6, or 8 h at room temperature before being placed in RNAlater and stored overnight at 4°C, after which they were frozen at -80°C. To investigate the effects of freeze-thaw cycling, HCC tumor specimens from 6 of the same patients used in the cold ischemia study were divided, preserved with RNAlater (within 30 min of excision) and subjected to 0, 1, 2, 4, 6 and 8 freeze-thaw cycles consisting of 1 h at room temperature and freezing at -80°C. To investigate the effects of the duration of frozen storage, the authors used retrospectively collected specimens from 96 patients that had been stored at -80°C for 0, 2, 4, 6, 8 or 10 y (16/timepoint). RNA was extracted using Trizol reagent and was evaluated using the Agilent RNA 6000 Nano kit on a bioanalyzer.  

   Summary of Findings:

   Mean RINs were comparable among HCC tumor and normal adjacent specimens (8.79 ± 0.43 versus 8.71±0.38, P=0.423).  RIN decreased progressively with cold ischemia time at a rate of 0.196 units/h in tumor and 0.193 units/ h in normal adjacent specimens. Significant differences inmean RIN, relative to control specimens placed in RNAlater within 30 min of excision, were observed in case-matched tumor specimens after a cold ischemia time ≥6 h (P=0.015 at 6 h and P=0.012 at 8 h) and in case-matched normal adjacent specimens after a cold ischemia time ≥2 h (P-0.012, P=0.007, P=0.003, and P=0.005 for 2, 4, 6, and 8 h cold ischemia time, respectively).  RIN also progressively declined with each additional freeze-thaw cycle at a rate of 0.148 units/cycle. A significant difference in mean RIN was noted in HCC tumor specimens that experienced 6 or 8 freeze-thaw cycles compared to those that were never frozen (P=0.005 and P=0.040, respectively). RIN also declined with frozen storage (-80°C) at a rate of 0.275 units/ year. Significantly lower mean RINs were found in HCC tumor stored for 6, 8 or 10 years compared to those stored for 0 years (P<0.001 for all). RIN was not significantly associated with patient age, gender clinical stage, tumor location, HBV infection, tumor grade, tumor diameter, or surgical approach using the Mann–Whitney U-tests/Kruskal– Wallis tests, but multivariate logistic regression using a binary RIN (threshold set to 8) identified a negative association between tumor grade and RIN (grade 3 versus grade 1-2, odds ratio of 0.08, P=0.014).

   Biospecimens

   • Tissue - Liver

   Preservative Types

   • RNAlater
   • Frozen

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Preaquisition	Prognostic factor	Tumor Stage I Tumor Stage II Tumor Stage III or IV Grade I or II Grade III Tumor diameter < 5 cm Tumor diameter 5-10 cm Tumor diameter >10 cm
   Preaquisition	Surgical procedure type	Open Colectomy Laparoscopic colectomy
   Preaquisition	Patient age	<50 years >50 years
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 4 cycles 6 cycles 8 cycles
   Biospecimen Acquisition	Cold ischemia time	<30 min 1 h 2 h 4 h 6 h 8 h
   Preaquisition	Diagnosis/ patient condition	HBV+ HBV-
   Biospecimen Acquisition	Biospecimen location	Left Right
   Storage	Storage duration	0 years 2 years 4 years 6 years 8 years 10 years
   Biospecimen Acquisition	Method of tissue acquisition	Laproscopic surgery Open surgery

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