In most cases, blood sample collection and further nucleic acid extraction from blood are conducted by different people at different places.
So, the storage duration and storage temperature, from blood collection to nucleic acid extraction, are critical to high-quality DNA and RNA samples.
Although the fresher the better is the golden rule when dealing with clinical samples, including liquid biopsies, we are interested in how much storage duration and storage temperature of blood affect the qualities of DNA and RNA. Therefore, we enrolled subjects and collected blood samples. These blood samples were stored for different durations and at different temperatures, followed by extracting nucleic acids and evaluating the qualities and quantities of DNA and RNA samples.
All subjects signed the informed consent. All samples were categorized according to storage duration and storage temperature. After specified storage duration at specific temperature, blood samples were treated with dextran to induce RBC aggregation. The supernatant was then harvested and RBC lysis buffer was added into supernatant.
After centrifugation, total WBCs were harvested from the pellets. The sequences of the primers used were as follow:. The RT master mix was prepared as follow: 0. The real-time PCR master mix was prepared as follow: 7. DNA methylation was assessed by pyrosequencing. The PCR reagent was prepared as follow: DNA samples ug were added to denaturation buffer 0. Then, samples were chilled on ice for 5min and applied to a positive charged nylon membrane Roche, Basel, Switzerland.
Membranes were probed with anti-5mC antibody , Cat. Subsequently, membranes were probed with either a rabbit or mouse IgG antibody conjugated to HRP for 1 hour at room temperature. Finally, the membranes were stained with Methylene Blue as loading control. In this study, statistical analyses were conducted with Microsoft Excel.
All quatitative raw data for plotting each figure and sub-figure is available in S1 Table. According to Fig 1A , if NGS and microarray experiments are expected, the blood samples can be stored, at room temperature, no longer than 24 and 32 hours, respectively. The samples in FR set were stored at freezer immediately after blood collection. Low temperature protected RNA from degrading at the duration of 40hr.
Each subject contributed three independent blood samples. From the preliminary result, 32hr is a clear duration point at which RIN value started to decline. To derive a more robust and unbiased conclusion, we conducted more detailed assays with more samples. In addition, when samples were stored at low temperature, the RNA quality remained without gradual declines. For the frozen samples, the RNA quality declined dramatically.
RNA samples are usually used for gene expression profiling with real-time quantitative polymerase chain reaction qPCR. Therefore, in addition to RNA quality examination, we also investigated the expression abundances on three common internal control genes with qPCR assay. When the samples were stored longer than 24 hours, the Ct values of U6 increased up to approximately 27 with little variations.
The Ct values of U6 and 18S significantly rose at the duration of 32hr. Compared with room temperature, low temperature did not protect genes from further degradation.
Fig 2B also showed that at the duration point 24, 32 and 40 hour, storage at low temperature made no difference on the Ct values of U6 compared from the one at room temperature. Such results implied that low temperature storage was also harmful to the expression consistency of 18S and GAPDH but harmless to the one of U6.
In summary, storage of whole blood in low temperature is not recommended. In addition to internal control genes, we also examined one chemokine gene, Interferon gamma-induced protein 10 IP, also named CXCL IP is induced by interferon gamma and involved in many inflammatory diseases such as chronic inflammatory arthritis, autoimmune diseases, type 1 diabetes, cardiovascular disease, psoriasis…etc [ 12 — 16 ].
Fig 2E showed that higher expression abundance of IP is expected in longer storage durations. When stored in tube, blood cells are quarantined from continuous supply of oxygen, nutrition and so on, which brought stress to blood cells.
In spite of the contradiction, our finding suggested that the blood samples should be stored for no longer than 24 hours at room temperature to obtain the best quality of RNA samples. To examine how much time can blood samples be stored without a significant decline in DNA quality, we first drew blood samples from one single subject, storing blood samples at room temperature and at different durations.
S2 Fig showed that the DNA samples with different durations aggregated with the size larger than 10K base pair. So, even stored for 15 days, DNA samples did not significantly degrade into small fragments. The same conclusion was also confirmed with Bioanalyzer DNA chip assay data not shown.
Since it seemed that DNA quality did not significantly decline with long storage duration, we turned to investigate DNA quantity, measuring the DNA concentration on the samples. Fig 3A demonstrated significant declines in DNA concentration for storage duration after 3. So far, our result showed that, with long storage duration, the quality of DNA did not decline but the concentration of DNA did. For an unbiased answer to this question, we drew 21 blood samples from each of the 10 subjects and repeated the analyses at four storage durations and at three temperatures.
Fig 3B showed that longer storage duration resulted in fewer WBC counts at both room and low temperature. In addition, for the same storage duration, low temperature also led to fewer WBC counts not illustrated in Fig 3B for simplicity. Such decline in WBC count should be resulted from cell lysis triggered by stress. For example, D0 denoted that whole blood was processed immediately after collected; D7 denoted that whole blood was stored for 7 days before further processing.
Further, we investigated DNA concentration. As shown in Fig 3C , at room and low temperature, longer storage duration also caused lower DNA concentrations.
Fig 3D showed that the two factors were highly correlated. Storing blood samples in tube stops the continuous supply of oxygen, nutrition and so on, which brought stress to blood cells. These stresses triggered blood cell lysis, lowering down the number of harvested WBC. Therefore, in addition to DNA quantity, we are also interested in whether storage duration and storage temperature affect DNA methylation.
To answer this question, we used pyro-sequencing facility Pyromark Q24 to specifically evaluate the methylation status of CpG marker cg located at the promoter region of CCL Each additive affects blood sample testing and storage in different ways.
For example, hematology procedures often require the blood to remain in the tube until the anticoagulants stabilize. Depending on the sample use, one of three temperatures will typically be specified for blood sample storage: room temperature, refrigerated, or frozen. Blood used for certain molecular genetic tests can remain stable for many days, with a wide range of acceptable temperature. DNA remains stable at room temperature for up to a month, but because live blood cells begin dying within two days, samples should be cultured or frozen in liquid nitrogen for future use.
Blood banks consider six weeks to be the "shelf life" of blood, but a study from Johns Hopkins University has shown that after three weeks, red blood cells are less effective at delivering oxygen-rich cells throughout the body. Blood stored longer than three weeks becomes less flexible and less able to fit in the body's smallest capillaries.
Depending on the blood's future use, longer storage without refrigerated or frozen temperatures can jeopardize its viability. For example, if stored blood is used in a transfusion, the blood never regains the flexibility that it lost after the three-week mark unrefrigerated in storage. Given the relatively rapid degradation of blood after it's drawn, all samples should be promptly tested, refrigerated for short-term storage, and frozen for long-term storage. Microsampling technology opens the door for new efficiencies in blood collection, shipping, and storage, including drying under ambient conditions and remote sampling.
Check out some complimentary blood microsampling resources to learn more, or explore our product line if you're ready for something new. TP was no difference up to 24 h. ALB had stable results up to 24 h. DBIL were stable both for 24 h. S23 , so as TC Fig. S24 and TG Fig. S26 and BUN exhibited differences 3 h. Egger test was applied to test publication bias. By trim and fill method, both the results of fixed and random effects model are just the same with original result Appendix 2, Fig.
S28 for funnel plot for trim-and-fill method. In the pre-analytic phase, reliable specimen storage is fundamental to high-quality test results Narayanan, Inappropriate storage conditions would pose a tangible challenge for the sample quality Adcock et al.
Significant time-and temperature-dependent changes can occur when the storage of blood is prolonged Hedberg and Lehto, ; Jobes et al. Earlier studies have reported acceptable stability after 24 h. More recently, different authors have reported that some measurements are stable up to 72 h. Storage time and temperature may have a small influence on WBC count. Although it hadn't analyzed in our study, there were studies reflect that WBC differential count was not stable over time Hill et al.
Although one study reported a better stability of the PLt count at room temperature Imeri et al. Another parameter reflects the propriety of platelet is MPV. From our results, it changed at the first compared point-in-time 1 h. The reliable MPV might have something to do with time- and concentration-related changes in platelet shape from discoid to spherical and swelling.
Those raise an important concern that refrigeration of specimens may not be satisfactory as previously believed.
As the time gone, RBC has been shown to significantly drop because of hemolysis. Parameters of CMP should also be considered for the time- and temperature- dependent change, although studies focused on this was relatively few. All in all, the reasons that may be responsible for change are as follows: firstly, self-consumption. What we could see was that even by 1 h. Nevertheless, our results showed that a sharp increase of blood potassium had occurred at the first hour under RT.
Whether refrigerator storage made a difference, requires more clinical trials. Thirdly, influenced by environment factors. TBIL was a parameter increased by hemolysis and decreased by longtime exposure under sunshine, so it is not stable and changes at 3 h. DBIL was relatively stable for 24 h.
BUN was another index influenced by exposure, as a result, it changed even at 3 h. ALB is an important part of plasma colloid osmotic pressure and was stable for 24 h. To our knowledge, this meta-analysis is the first study which systematically estimates the effect of storage conditions on CBC and CMP testing and identified that time and temperature of storage can indeed have an impact on the quality of testing.
The most important implication of this study is the need to define reliable time and means of sample storage, help establish of centralized hematological services or biobanks and benefit transfusion. WBC count was relatively stable and the results had no significant change up to 3 d under RT. Interestingly, at some time-points 1, 2 and 4 hr. MPV was not a very stable measurement for samples stored over time or temperature.
It changed at the first compared time 1 hr. At 2 hr. Even just 12 hr. HGB comparison of 1 hr. Except for 8 hr. HCT was also a parameter that changed approximately at 8 hr. Even the sample was stored for only one hour at RT, the stability was unsatisfactory.
The results of Na changed at 12 hr. For 12 hr. Samples for AST had no difference for 24 hr. Samples for ALT had no difference for 24 hr. TP was no difference up to 24 hr. ALB had stable results up to 24 hr. We are grateful to David R.
Dong-wen Wu: designed the research; searched the lecture; wrote the paper. Yu-meng Li: screened and evaluated the quality of evidence; extracted data; helped write the paper. Fen Wang: screened and evaluated the quality of evidence; extracted data. National Center for Biotechnology Information , U. Journal List EBioMedicine v. Published online Sep Author information Article notes Copyright and License information Disclaimer.
Dong-wen Wu: nc. This article has been cited by other articles in PMC. Associated Data Supplementary Materials Supplementary material. Results A total of 89 studies were confirmed. Introduction Delayed sample analysis for organizational, technical reasons or questionable results that need to be verified are not rare in clinical practice Lippi and Simundic,
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