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Effects of storage temperature, storage time, and hemolysis on the RNA quality of blood specimens: A systematic quantitative assessment.

Author(s): Jiang Z, Lu Y, Shi M, Li H, Duan J, Huang J

Publication: Heliyon, 2023, Vol. 9, Page e16234

PubMed ID: 37260878 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared RNA yield, purity, integrity and expression among blood specimens stored at room temperature for up to 24 h and at 4°C for 1 week with matched specimens that were not stored before analysis. Additionally, RNA yield, purity, integrity and expression were compared between hemolyzed blood specimens that were and control specimens (non-hemolyzed). The authors used RNA quality number (RQN) and the ratio of 28S to 18S ribosomal RNA (28S/18S) to evaluate the RNA integrity of specimens and surplus blood specimens. EDTA blood was collected from 6 healthy volunteers and aliquoted. Matched aliquots of volunteer blood were stored at room temperature (22–30°C) for 1 h, 2 h, 6 h, 12 h, and 24 h. Matched aliquots of four pediatric blood specimens were stored at 4°C for 2 h, 6 h, 12 h, 24 h, 48 h, 3 days, and 1 week.  Hemolyzed blood specimens included 6 specimens that were collected in the department that did not meet laboratory test standards and 6 specimens obtained by freezing blood at -80°C for 30 min and thawing at room temperature for 30 min.  Blood cells were obtained by centrifugation at 500 g for 10 min at 4°C. Hemolysis was analyzed using the Free Hemoglobin Assay Kit on a spectrophotometer. RNA was isolated using RNA Simple Total RNA Kit and quantified by spectrophotometer. RNA integrity was evaluated using the Bioptic Qsep 100 Capillary Electrophoresis System. 18S rRNA (18S), β-actin (ACTB), hypoxia-inducible factor-1 α (HIF1α), heme oxygenase 1 (HMOX1), and marker of proliferation Ki-67 (MKI67) were quantified by real-time RT-PCR and normalized to glyceraldehydes-3-phosphate dehydrogenase (GAPDH).

   Summary of Findings:

   RNA yield and purity (ratio of absorbance at 260 nm to 280 nm) were unaffected by blood storage at room temperature for up to 24 h. However, both markers of RNA integrity (RQN and 28S/18S) declined significantly with storage at room temperature for 6 h compared to 0 h controls (P=0.002 and P<0.001, respectively).  RQN was strongly and negatively correlated with blood storage duration at room temperature (r=-0.787, P<0.0001). Similarly, while storage at 4°C did not affect RNA yield or purity of pediatric blood specimens, RQN was significantly lower in specimens stored for 48 h compared to specimens stored for 2 h (P=0.048), and both RQN and 28S/18S ratio were significantly lower in blood stored for 1 week compared to blood stored for  2 h (P=0.001, for both). RQN was strongly and negatively correlated with blood storage duration at 4°C (r=-0.863, P<0.0001), but remained in the normal range (>7) in specimens stored for ≤ 72 h. RNA concentration and purity were unaffected by freeze-thaw induced hemolysis, but both RQN and 28S/18S ratio were significantly lower in freeze-thaw cycled specimens (P=0.005 and P=0.0001, respectively). In contrast, routinely collected hemolyzed specimens had comparable RQN to control specimens, but still had a lower ratio of 28S to 18S rRNA (P=0.007) and a higher hemoglobin concentration (P=0.002). Importantly, when white blood cells were mixed with freeze-thawed RBCs RNA integrity remained high but when the freeze-thaw white blood cells were added to RBCs the integrity was significantly lower (P<0.05 and P<0.01, respectively). The authors state this indicates that leukocyte lysis and not RBC lysis is responsible for the reduction in RNA integrity. Analysis of surplus laboratory blood specimens found that 33.33% had an RQN <7 and 23.33% had a 28S/18S ratio < 1, indicating the specimens had been stored too long.

   Effects of storage on mRNA levels were transcript-specific and storage temperature- and duration-dependent. Compared to blood analyzed immediately, blood stored at room temperature for 2 h had higher levels of MK167 (P<0.001) and HIF1α (P<0.01), blood stored for 6 h had lower levels of HIF1α (P<0.01) and HMOX1 (P<0.001), blood stored for 12 h had lower HIF1α (P<0.01), and blood stored for 24 h had lower ACTB levels (P<0.01).  Compared to unstored blood, blood stored at 4°C  for 2 h had lower levels of 18S rRNA (P<0.001); blood stored for 6 h had lower levels of 18S rRNA (P<0.001), ACTB (P<0.001) and MK167 (P<0.001); blood stored for 12 h had lower HIF1α (P<0.001) and MK167 (P<0.001), blood stored for 24 h had lower HIF1α (P<0.001), and blood stored for 72 h had higher ACTB levels (P<0.01). Freeze-thaw hemolyzed blood had lower ACTB (P<0.05), HIF1α (P<0.01), MK167 (P<0.05) levels than control non-hemolyzed blood.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Biospecimen Aliquots and Components	Hemolysis	Freeze/thaw-induced Present Absent
   Storage	Freeze/thaw cycling	0 cycles 1 cycle
   Storage	Time at room temperature	1 h 2 h 6 h 12 h 24 h 0 h
   Storage	Storage duration	2 h 6 h 12 h 24 h 48 h 3 days 1 week 0 h

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