Which of the following analytes would be increased due to delay in centrifugation?

As part of a study designed to assess the nutritional status of astronauts during long-duration space flight, in-flight phlebotomy sessions are performed on the International Space Station (ISS). Blood specimens are nominally centrifuged within 45 min of phlebotomy, and then stored at − 80 °C. Because of the nature of the space missions, blood processing can unavoidably be delayed beyond the standard recommended 30- to 120-min timeframe. This recommended timeframe is based on data showing that changes in glucose, potassium, and lactate dehydrogenase occurred after 120 min of storage of whole blood at room temperature.

Prompt centrifugation is critical to preserve the integrity of blood samples when measuring certain analytes, including cholesterol, creatinine, potassium, calcium, and chloride [1], [2], [3], [4]. Other analytes, such as triglycerides, plasma parathyroid hormone (PTH), sodium, and ferritin [5], [6], [7], have been shown to be unaffected by increased storage time (up to 48 h) before centrifugation. Prolonged contact of serum or plasma with red blood cells can result in an exchange of substances between serum and the cells, which can increase or decrease analyte concentration in serum. Hemolysis can result in altered values for similar reasons.

To our knowledge, no studies have been done to investigate the effects of delayed centrifugation on other markers of bone metabolism and clinical chemistry, including bone-specific alkaline phosphatase (BSAP), osteocalcin, and vitamin D.

In this study, we sought to determine the effects of delayed centrifugation on the concentrations of 36 analytes nominally measured during space flight as part of the nutritional status assessment experiment. The results presented here are data from a ground-based study designed to parallel the conditions on the ISS that could lead to delayed centrifugation. These data establish information about the integrity of samples that undergo delayed processing up to 24 h after phlebotomy.

Subjects

Eleven healthy subjects were recruited for the study. Five were recruited for the initial study and then 6 subjects were added to the study 1 y later. The findings from the initial study with 5 subjects indicated that meaningful results could be obtained by having more subjects but analyzing at fewer time points for some of the tests. The following analytes were measured 45–60 min and 24 h after phlebotomy: 25-hydroxyvitamin D, calcium, bone-specific alkaline phosphatase, retinol-binding

Results

After 24 h, the blood tubes looked as if the serum and cells had separated somewhat. No hemolysis was visible after 2.5, 5, or 24 h.

Table 1 lists the mean concentration (for all subjects) and standard deviation of each analyte at each time point after phlebotomy. Five of the analytes tested – PTH, osteocalcin, zinc, pyridoxal 5′-phosphate (PLP), and homocysteine – showed statistically significant changes in mean concentration after delayed centrifugation. Fig. 1 shows the changes in mean

Discussion

The results from this study not only affect the use of blood samples in nutritional status assessment of astronauts on the International Space Station, but also have wide application to ground-based situations. In a hospital or field location, a blood sample may not be processed immediately because of the logistics of the situation.

The present study demonstrates that 25-hydroxyvitamin D, calcium, alkaline phosphatase, BSAP, retinol-binding protein, ceruloplasmin, cortisol, total antioxidant

Acknowledgments

Funding support: This research was supported by the National Aeronautics and Space Administration's Human Research Program.

Financial disclosures: None declared.

We thank Jane Krauhs of Wyle Integrated Science and Engineering Group for editing the manuscript.

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  Some reports included data at different times and temperatures; studies are generally dedicated to one specific discipline; the number of samples in analyte studies is sometimes very low [9,10]; samples are centrifuged and subsequently aliquoted and frozen before testing which can introduce a bias into the results [5,11]. In these studies, the observed variations were analyzed in different ways: by statistical analysis such as Student's t-test [5], ANOVA [12], Wilcoxon [13], the Bonferroni t test [10], a change more than 10% relative to baseline concentrations [11,14] or allowable variability as given by the German Federal Medical Council recommendations [8], or from an analytical SCL (significant change limits) approach [15] or combined analytical and biological variation [9]. These various methods give considerably different performance limits [16].

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