Stability of plasma analytes after delayed separation of whole blood: implications for epidemiological studies

Sarah Clark1, Linda D Youngman1,2, Alison Palmer1, Sarah Parish1, Richard Peto1 and Rory Collins1

1 Clinical Trial Service Unit & Epidemiological Studies Unit, Nuffield Department of Clinical Medicine, University of Oxford, UK.
2 Current affiliation: FDA Office of Research, Laurel, MD 20708, USA.


    Abstract
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Background Large blood-based epidemiological studies require simple, cost-effective sample collection methods. Immediate sample separation or rapid transport of chilled blood samples to a central laboratory may be impractical or prohibitively expensive. To assess the feasibility and reliability of transporting blood samples over several days at ambient temperature (e.g. by mail), we evaluated the stability of various plasma analytes in samples stored at room temperature or chilled.

Methods Multiple vacutainers of blood, containing EDTA and aprotinin as preservative, were drawn from 12 volunteers and stored at 21°C or 4°C. Immediately after collection and 1, 2, 3, 4, and 7 days later, vacutainers stored at each temperature were centrifuged, and the plasma was aliquoted and stored at –80°C. Subsequently, all aliquots from each individual were analysed in one analytical run for a range of chemistries.

Results In whole blood stored at room temperature for up to 7 days, concentrations of albumin, apolipoproteins A1 and B (apoA1 and apoB), cholesterol, high density lipoprotein (HDL), total protein, and triglycerides changed by less than 4%, and low density lipoprotein (LDL) by less than 7%. Whilst alanine transaminase (ALT), creatine kinase (CK), creatinine, and {gamma}-glutamyl transferase (GGT) concentrations changed substantially at room temperature, there was less than 4% change during chilled storage up to 7 days. By contrast, aspartate transaminase (AST) concentrations increased markedly under both conditions.

Conclusions A wide range of important analytes, including lipids, change by only a few per cent in whole blood during storage at room temperature for several days. Mailed transport of whole blood samples may, therefore, be a simple and cost-effective option for large-scale epidemiological studies.


Keywords Analytes, blood-based epidemiology, blood collection, stability, temperature

Accepted 3 October 2002

Observational epidemiological studies can often be greatly enhanced by the inclusion of biochemical analyses in stored blood samples collected from the population being studied. Biochemical analyses can be used to assess risk factor exposure, to control for confounding, or to measure the effects of bias. In randomized trials, biochemical analyses can be used to monitor the safety and biochemical efficacy of treatment. For epidemiological studies to be informative, they often need to be large (perhaps involving tens or hundreds of thousands of individuals), which in turn requires methods for blood collection that are practical and cost-effective. Standard guidelines for blood sample handling state that plasma or serum should be separated from cells as soon as possible and certainly within2 hours.1 Whilst this is necessary for particular analytes, it might be assumed that many blood analytes deteriorate within a matter of hours in unseparated samples kept at ambient temperature. This perceived need for either immediate local separation of blood samples or their rapid chilled transfer toa central laboratory, which increases complexity and cost, can prevent blood collection from being included in large-scale epidemiological studies.

Particularly when blood samples are being collected in a large number of separate sites within a study, mailing of whole blood samples to a central laboratory for separation may be more convenient and cost-effective than making arrangements for local separation or for courier transport of chilled samples. Saliva samples have been successfully collected by mail for measurement of hepatitis B virus seropositivity2 and the potential advantages of collecting blood samples by mail in epidemiological studies have been noted.3 There is limited evidence to suggest that some biochemical analytes (such as total cholesterol) may be stable in whole blood for several days at ambient temperature.4–6 If confirmed for a wider range of analytes of interest, use of mailed whole blood samples might allow blood collection to be included in studies where it would not otherwise be considered feasible. We have evaluated the use of mailed whole blood samples and of chilled samples (compared with immediate plasma separation) by simulating these two collection methods in the laboratory, and assessing their impact on the stability of a range of blood analytes.


    Materials and Methods
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Subjects and procedures
Twelve 10 ml vacutainers of blood containing 0.12 ml preservative (15% potassium EDTA with aprotinin 0.34 mmol/l [Becton Dickinson UK Limited, Oxford, England]) were drawn from each of 12 non-fasting volunteers (5 males and 7 females, aged 25–60 years). Each vacutainer corresponded to a particular storage temperature (room temperature or chilled) and separation time-point (immediate or 1, 2, 3, 4, or 7 days post-collection), which were randomly assigned within each volunteer’s set of vacutainers using a random number table.

Two of the 12 vacutainers from each individual were centrifuged immediately after collection (2100 g for 15 minutes at 4°C), and the plasma was aliquoted into 1.8 ml Nunc cryotubes (Nunc A/S, Roskilde, Denmark) and stored at –80°C. The remaining 10 vacutainers from each individual were covered in aluminium foil to prevent any potential effect from light (since samples would be unlikely to be exposed to light during transportation) and kept at room temperature (defined as 21°C) or chilled (4°C). At each subsequent time-point, a vacutainer from each temperature for each volunteer was retrieved, centrifuged and the plasma aliquoted and stored at –80°C. (An on-going study in our laboratory has found no change in concentrations of any of the analytes in plasma samples stored at –80°C during up to 5 years.)

Biochemical analyses
Prior to analysis, the frozen samples were left to stand at room temperature to thaw, then inverted several times to mix. The plasma aliquots from all temperature and time-points for each volunteer were analysed together in one batch, to avoid run-torun variability, for the following analytes: alanine transaminase (ALT), albumin (by the bromocresol purple method), apolipoproteins A1 and B (apoA1 and apoB), aspartate transaminase (AST), creatine kinase (CK), creatinine (by the Jaffe rate method), {gamma}-glutamyl transferase (GGT), high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol, total protein (by the rate biuret method), and triglycerides. Measurements were performed on a Synchron LX20 autoanalyser (Beckman Coulter UK Limited, High Wycombe, England), using Beckman Coulter reagents, calibrators, and controls for all assays, except for HDL and LDL which were analysed using N-geneous reagents, calibrators, and controls (Bio-Stat Limited, Stockport, England). These direct methods for quantification of HDL and LDL were fully automated, involving chemical isolation of the lipoprotein and enzymatic measurement of cholesterol. The Synchron LX20 autoanalyser monitors absorbance readings at multiple wavelengths and was programmed to subtract a sample blank absorbance reading from the final reaction absorbance where possible.7 This helps to correct for interference from colour in plasma due to haemolysis, which was a significant problem in samples from the later time-points. The intra-assay coefficient of variation (CV) for ALT was 4% at a quality control level of 24.0 U/l and 1% at a quality control level of 84.3 U/l; for albumin was 1% at 29.3 g/l and 2% at 47.2 g/l; for apoA1 was 3% both at 83.9 mg/dl and 279.7 mg/dl; for apoB was 4% both at 95.7 mg/dl and 200.9 mg/dl; for AST was 3% at 29.8 U/l and 1% at 100.4 U/l; for CK was 3% at 56.9 U/l and 1% at 394.0 U/l; for creatinine was 9% at 73.6 µmol/l and 3% at 325.1 µmol/l; for GGT was 7% at 20.1 U/l and 3% at 107.4 U/l; for HDL was 5% both at 0.7 mmol/l and 2.2 mmol/l; for LDL was 5% both at 2.5 mmol/l and 5.6 mmol/l; for cholesterol was 2% both at 4.3 mmol/l and 9.9 mmol/l; for total protein was 1% both at 45.7 g/l and 73.7 g/l; for triglyceride was 3% both at 2.0 mmol/l and 4.7 mmol/l.

Data analysis
For each individual, the mean analyte concentration from analysis of the two samples that had been separated and frozen immediately was used as the baseline ‘fresh’ value against which analyte concentrations at future time-points were compared. The stability of an analyte under each temperature condition was determined by calculating the percentage change in concentration from the mean fresh value at each time-point for each individual, then calculating the mean percentage change from fresh (and standard error of the mean) at each time-point from the individual data. Log-linear regression, incorporating a term for individual, was used to determine the percentage change per day for each analyte in samples kept at room temperature and chilled; to test for significant differences in analyte concentrations between storage temperatures; and to test for significant trends over time at each temperature. Data analysis was performed using SAS (SAS Institute Inc, Cary, NC, USA).


    Results
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 Materials and Methods
 Results
 Discussion
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Table 1Go shows the results obtained in samples separated immediately after collection from the volunteers, along with the reference intervals for each analyte. On the whole, the results obtained for each analyte give good coverage of the reference interval, with some results being outside of this range.


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Table 1 Assay values in samples separated immediately after collection from 12 healthy volunteers aged 25–60 years (with reference intervals)
 
Table 2Go shows the mean percentage change in plasma analyte concentrations at each time-point, for samples stored at room temperature (21°C) and chilled (4°C). Figure 1Go summarizes the data by showing the mean percentage change per day for each analyte under each temperature condition. At 21°C, the concentrations of albumin, apoA1, apoB, total cholesterol, HDL, total protein, and triglycerides differed by less than 4% from the fresh value up to 7 days after blood collection, while the concentration of LDL differed by less than 7%. The mean percentage change per day at room temperature was less than 0.5% for all of those analytes, except for HDL and LDL where it was less than 1%. With the exception of total cholesterol, the change over time did not differ significantly between samples stored at 21°C or 4°C. For total cholesterol, there was a significant difference (P = 0.0003) between the values in samples kept at the different temperatures, but it was small and did not become apparent until 7 days.


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Table 2 Percentage change in analyte concentration over time in samples stored at 21°C and 4°C
 


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Figure 1 Percentage change per day (95% CI) for analytes in whole blood samples stored at 21°C or 4°C for up to 7 days

 
By contrast, concentrations of ALT, CK, creatinine, and GGT changed more markedly at 21°C: for example, creatinine increased by more than 20% in whole blood after 3 days. Under chilled conditions at 4°C, however, these analytes were more stable, and changed by less than 4% in whole blood after 7 days (P < 0.0001 for 21°C versus 4°C). The mean percentage change per day for these analytes under chilled conditions was less than 0.7%. Although a significant trend (P = 0.0008) towards an increase in CK values was observed over time in samples kept at 4°C, the change was small. Of the analytes measured, only AST was found to change substantially under both temperature conditions. The concentration of AST increased by more than 7% after 3 days at 4°C, and by more than 15% after just one day at 21°C. The percentage changes in ALT and AST appeared to be somewhat greater in the samples with lower baseline levels compared with higher baseline levels, but more individuals with a wider range of baseline values would need to be studied to assess this reliably.


    Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
The stability of a wide range of plasma analytes in whole blood samples stored for several days is much better than had perhaps been widely believed. The present study demonstrates that albumin, apoA1 and apoB, total cholesterol, HDL, LDL, protein, and triglycerides can be measured reliably in whole blood samples kept at room temperature for at least a week before plasma separation. Alanine transaminase, CK, creatinine, and GGT exhibited greater change at room temperature, hence reliable quantification of these analytes would require whole blood samples to be kept chilled before separation. However, since the mean concentrations of these analytes were found to change by less than 4% during up to 7 days in chilled blood, such samples do not require rapid transfer to a central laboratory for separation. The most unstable analyte was found to be AST, with reliable quantification of this analyte requiring blood samples to be separated immediately or, if kept chilled, within 24 to 48 hours.

Only three previous studies have investigated the stability of general clinical chemistry analytes in blood kept unseparated beyond 24 hours. Ono et al. drew blood from 10 volunteers into plain serum tubes and left aliquots of whole blood to stand at 4°C, 23°C, or 30°C for up to 48 hours.4 They found no significant change in albumin, total cholesterol, triglyceride, or total protein at 4°C or 23°C, which is in agreement with the present study. However, they also found GGT and creatinine to be stable at these temperatures, whereas we found them to change by 8% and 18% respectively after 48 hours at 21°C. Whilst Ono et al. found little change in ALT and AST at 4°C, the concentrations increased significantly after 8 hours at 23°C. We found ALT to change by only 2% up to 48 hours at 4°C and 21°C, but AST changed by 5% at 4°C and by 35% at 21°C after 48 hours. These discrepancies may reflect differences in the analytical methods used in the present study compared with those used 20 years ago by Ono et al., and the use of serum rather than plasma samples.

In another study, Hankinson et al.5 investigated the stability of total cholesterol, HDL, apoA1, and apoB in whole blood samples taken from 12 individuals and stored at room temperature (21°C) in ambient light for up to 72 hours, or placed in a Styrofoam mailer with a frozen gel pack (which maintained a temperature of 4°C for 20 hours) for up to 48 hours. The study design was similar to that of the present study, with whole blood samples stored under each temperature condition up to the appropriate time-point, then centrifuged, the plasma aliquoted and frozen at –80°C. Samples from all time-points were analysed in one analytical run. The concentrations of all of the lipids were found to change non-significantly in chilled blood. However, at room temperature, although total cholesterol and apoA1 changed only non-significantly, HDL and apoB increased by 3.0% and 5.2% per day respectively. By contrast, in the present study, HDL and apoB changed by –0.6% and +0.4% per day respectively at room temperature. The discrepancies between the results of the two studies may also reflect differences in the analytical methods. Hankinson et al. measured HDL by precipitation of very low density lipoprotein (VLDL) and LDL with dextran sulphate and magnesium chloride, apoA1 was measured in the HDL and HDL3 fractions by radial immunodiffusion and apoB measured in whole plasma by radial immunodiffusion. By contrast, in the present study, the analytes were measured directly in plasma by automated methods optimized to deal with potential interference from colour due to haemolysis, which becomes more of a problem as the delay to plasma separation increases.7 In addition, the blood samples stored at room temperature in the study by Hankinson et al. were exposed to ambient light, which might have affected stability of HDL and apoB, whereas samples in the present study were stored in the dark.

Finally, Heins et al. investigated the stability of various analytes in whole blood samples taken from 20 apparently healthy individuals into plain serum tubes and then stored chilled (9°C) or at room temperature (23–27°C) in the dark for 7 days.6 An analyte was described as unstable if the change in concentration was significantly greater than the maximum allowable inaccuracy according to the Guidelines of the German Federal Medical Council: 6% for creatinine, cholesterol, HDL, and LDL; 7% for AST, ALT, and GGT; and 8% for CK. These investigators reported ALT, AST, and cholesterol to be stable for 7 days at 9°C, and for 3 days at room temperature. By contrast, however, we found that the mean concentration of AST changed by more than 14% after 7 days under chilled conditions and by over 40% after 3 days at room temperature. Other studies have also found a substantial increase in AST, due to haemolysis and a 40-fold higher activity of AST in erythrocytes compared with serum.8,9 Heins et al. reported HDL, LDL, CK, and GGT to be stable for 7 days at 9°C, but not at 23–27°C. We also found CK to change by less than 8%, and GGT to change by less than 7%, after 7 days under chilled conditions. Moreover, in our study, HDL and LDL changed by less than 7% after 7 days not only under chilled conditions, but also at room temperature. Heins et al. found that creatinine concentrations increased by around 20% at room temperature over 3 days (which agrees with the present study) but decreased by around 8% over the same period at 9°C (whereas we found less than a 4% change over 7 days under chilled conditions). Discrepancies between the results of Heins et al. and the present study might be explained by differences in analytical methods, by the ways of dealing with interference from haemolysis, by differences in temperature conditions, or by the use of serum rather than plasma samples. Furthermore, Heins et al. performed biochemical measurements on the samples immediately after centrifugation at each time-point, rather than freezing the samples and analysing them all in the same run, as in the present study, so that day-to-day assay variability may have affected results.

Many analytes considered in the present study did exhibit a slight increase in concentration over time, which agrees with a previous study.7 This is contrary to what one might expect to observe from analyte degradation, and seems likely to be due to leakage of water from the plasma into the red cells (following failure of the sodium/potassium pump to maintain osmotic balance) resulting in swelling of red cells, increase in the haematocrit, decrease in the plasma volume and, therefore, increased concentration of plasma analytes. It has also been reported that certain analytes, particularly AST, have a much higher activity in red blood cells than plasma, causing an increased concentration in haemolysed plasma.8,9 Furthermore, it has been suggested that the increase in creatinine concentration during storage is due to non-specific formation of pseudocreatinines.6 Recording the length of time from collection to separation of each sample might allow appropriate adjustment to be made for the slight increase in concentration of these analytes over time. The degree of change in analyte concentrations may differ slightly in a real situation compared with a laboratory setting, as is suggested by some of the results from Hankinson et al.5 In a study involving mailed blood samples from around 20 000 individuals, cholesterol concentrations in samples that had spent 4 days in the post were an average of about 7% higher than in those that had spent one day in the post, but there was less than a 2% difference in apoA1 levels (Clark S, Youngman LD, Parish S, Palmer A, Peto R, Collins R. Total cholesterol, apolipoproteins B and A1, and non-fatal myocardial infarction: 19 594 male and female cases and controls of ISIS-3. Unpublished manuscript). It should also be noted that ‘room temperature’ in the present study was defined as 21°C, which may not be realistic for studies in hotter climates (and further stability studies under such conditions need to be conducted). However, since many analytes were found to change by only a few per cent over at least 7 days at 21°C, it might be supposed that any change at higher temperatures may remain in this range for at least 3 to 4 days, which might still be a feasible time-frame within which to transport whole blood samples to a central facility.

In conclusion, we have shown that the concentrations of many analytes change by only a few per cent in whole blood stored at room temperature for up to 7 days, and may therefore be measured reliably in mailed blood samples. These results are relevant for planning blood-based epidemiological studies. Depending upon the analytes to be measured, blood collection methods could be greatly simplified and, hence, the costs vastly reduced, enabling blood collection to be included in studies where it would not otherwise be considered feasible.


KEY MESSAGES

  • Simple blood collection methods could help make large blood-based epidemiological studies feasible.
  • The concentrations of a wide range of analytes (including lipids) may be measured reliably in whole blood stored at room temperature for up to 7 days.
  • Collection of whole blood samples by mail is a simple and cost-effective way to facilitate the inclusion of blood assays in large-scale epidemiological studies.

 


    Acknowledgments
 
This work was supported by the Medical Research Council, the British Heart Foundation and Cancer Research UK. We gratefully acknowledge Mary Bradley, Tatyana Chavagnon, Buki Chukwurah, Kathy Emmens, Joanne Gordon, Joy Hill, Meng Jie Ji, Karen Kourellias, Stuart Norris, Leon Peto, Martin Radley, Janet Taylor, Jane Wintour, and Marie Yeung of the Clinical Trial Service Unit and Epidemiological Studies Unit (CTSU) laboratories. Dr Robert Clarke provided helpful comments on the manuscript.


    References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
1 Young DS, Bermes EW. Specimen collection and processing: sources of biological variation. In: Burtis CA, Ashwood ER (eds). Tietz Textbook of Clinical Chemistry. Philadelphia: WB Saunders Company, 1999, pp. 42–72.

2 O’Connell T, Thornton L, O’Flanagan D et al. Oral fluid collection by post for viral antibody testing. Int J Epidemiol 2001;30:298–301.[Abstract/Free Full Text]

3 Perneger TV, Etter J-F. Commentary: Extending the boundaries of data collection by mail. Int J Epidemiol 2001;30:301–02.[Free Full Text]

4 Ono T, Kitaguchi K, Takehara M, Shiiba M, Hayami K. Serum-constituents analyses: effect of duration and temperature of storage of clotted blood. Clin Chem 1981;27:35–38.[Abstract]

5 Hankinson SE, London SJ, Chute CG et al. Effect of transport conditions on the stability of biochemical markers in blood. Clin Chem 1989;35:2313–16.[Abstract]

6 Heins M, Heil W, Withold W. Storage of serum or whole blood samples? Effects of time and temperature on 22 serum analytes. Eur J Clin Chem Clin Biochem 1995;33:231–38.[ISI][Medline]

7 Youngman LD, Lyon VE, Collins R et al. Problems with mailed whole blood in large-scale epidemiological studies and methods of correction. (Abstract). The FASEB Journal 1993;1:377.

8 Sonntag O. Haemolysis as an interference factor in clinical chemistry. J Clin Chem Clin Biochem 1986;24:127–39.[ISI][Medline]

9 Frank JJ, Bermes EW, Bickel MJ et al. Effect of in vitro hemolysis on chemical values for serum. Clin Chem 1978;24:1966–70.[Abstract]