Evaluation of a reduced centrifugation time and higher centrifugal force on various general chemistry and immunochemistry analytes in plasma and serum

Abstract

Background

Centrifugation of blood samples is an essential preanalytical step in the clinical biochemistry laboratory. Centrifugation settings are often altered to optimize sample flow and turnaround time. Few studies have addressed the effect of altering centrifugation settings on analytical quality, and almost all studies have been done using collection tubes with gel separator.

Methods

In this study, we compared a centrifugation time of 5 min at 3000 × g to a standard protocol of 10 min at 2200 × g. Nine selected general chemistry and immunochemistry analytes and interference indices were studied in lithium heparin plasma tubes and serum tubes without gel separator. Results were evaluated using mean bias, difference plots and coefficient of variation, compared with maximum allowable bias and coefficient of variation used in laboratory routine quality control.

Results

For all analytes except lactate dehydrogenase, the results were within the predefined acceptance criteria, indicating that the analytical quality was not compromised. Lactate dehydrogenase showed higher values after centrifugation for 5 min at 3000 × g, mean bias was 6.3 ± 2.2% and the coefficient of variation was 5%.

Conclusions

We found that a centrifugation protocol of 5 min at 3000 × g can be used for the general chemistry and immunochemistry analytes studied, with the possible exception of lactate dehydrogenase, which requires further assessment.

Introduction

The centrifugation of blood samples to separate serum or plasma from platelets and blood cells is an important preanalytical activity and influences sample flow and turnaround time in the laboratory. Clinicians are often guided by laboratory tests in order to support clinical decision-making, and therefore desire rapid turnaround times from sample collection until test results are ready. Since centrifugation is a time-consuming step in sample processing, short centrifugation times are desirable as long as analytical quality is not compromised. Manufacturers of blood collection tubes typically recommend centrifugation for 10 to 15 min depending on the type of tube,^1,2 and WHO also, in general, proposes a centrifugation time of at least 10 min and 1500 × g for serum and at least 15 min and 2000–3000 × g for plasma. ³ Only a few scientific studies have investigated the influence of centrifugation settings of less than 10 min on laboratory results in serum or plasma,^4–9 and apart from one, all were performed with tubes containing a gel separator. A possible problem with the use of gel separator tubes is gel flotation in samples with a high concentration of protein, which can lead to mechanical disruption upon aspiration into instrument sampling probes. ¹⁰ In this study, we used plasma and serum tubes without a gel separator to investigate whether an accelerated centrifugation protocol of 5 min at 3000 × g can be applied for general chemistry and immunochemistry analytes without compromising their analytical quality.

Materials and methods

Sample collection and processing

Venous blood samples were collected by experienced phlebotomists from a total of 80 hospitalized patients, outpatients and healthy laboratory staff, who all gave their informed consent. Blood samples from patients were collected during a routine phlebotomy. From each individual, two pairs of samples were collected into four tubes in random order from one venepuncture. All samples were anonymized immediately after phlebotomy. Samples were collected into BD Vacutainer serum tubes (REF 369032) (n = 40) or BD Vacutainer lithium-heparin tubes (REF 368884) (n = 40) (Becton Dickinson, Plymouth, UK). Serum samples were allowed to clot for 30 min before processing. One pair of tubes was centrifuged for 10 min at 2200 × g (3370 r/min), according to laboratory routine practice, and the other pair was centrifuged for 5 min at 3000 × g (3935 r/min) (Figure 1).

Figure 1.

Flowchart of the study design showing sample collection and centrifugation. The four tubes sampled from one subject compose a sample set (e.g. tube 1 P, 2 P, 3 P and 4 P). The two tubes sampled from one subject and centrifuged using the same setting compose a sample pair (e.g. tube 1 P and 2 P).

Centrifugation was carried out using an Eppendorf 5810 R swing bucket centrifuge at a controlled temperature of 21 ± 1℃ and acceleration and braking set at step 9 corresponding to approximately 25 s. All samples were kept at room temperature and centrifuged within 1 h from sampling. Following centrifugation, all samples were inspected visually to look for any visible haemolysis, icterus and lipaemia and the quality of plasma/serum separation from the blood cells. They were then analysed immediately in random order. Following advice from The Central Denmark Region Committees on Health Research Ethics, we were informed that ethical approval was not required for this study.

Methods of analysis

In all tubes, the following selection of general chemistry and immunochemistry analytes was measured: cobalamin (B12), calcium (CA), folic acid (FOL), free triiodothyronine (FT3), immunoglobulin M (IgM), phosphorus (P), potassium (K), haemolysis index (HEM), icterus index (ICT) and lipaemia index (LIP). In addition, lactate dehydrogenase (LDH) was measured in lithium-heparin tubes, and total protein (TP) was measured in serum tubes. Single analysis was performed for all analytes. The above analytes were selected, because they are generally sensitive to haemolysis and preanalytical factors, and furthermore LDH, K and P have been shown to be appropriate for evaluating centrifuge settings. ¹¹ The sample materials used for the analytes in this study were based on laboratory routine procedures. All analytes, except TP, were measured in lithium-heparin plasma, since this is the primary sample material used in our laboratory. Additionally, analytes were measured in serum, since this is occasionally used as alternative material. Unfortunately, LDH was only measured in plasma. Sample tubes for TP are also used for electrophoresis, for which plasma is not considered suitable, and hence TP was only measured in serum. Analysis was performed using an Abbott Architect c16000 (CA, IgM, LDH, K, P, TP, HEM, ICT and LIP) and an Abbott Architect i2000SR (B12, FT3 and FOL), both from Abbott Diagnostics, Abbott Park, IL, USA.

Statistical analysis

Differences were calculated for each sample set as the mean of the sample pair centrifuged for 10 min at 2200 × g subtracted from the mean of the sample pair centrifuged for 5 min at 3000 × g, and these differences were depicted in difference plots using total error (TE) as acceptance limits. Total error was calculated as

$\text{TE}=1.65\times \text{CVgoal}+\text{biasgoal}$

Mean differences and 95% confidence intervals were calculated and compared with bias goals, which refer to limits for maximum allowable bias used in laboratory routine quality control.

For each of the two centrifugation settings (10 min at 2200 × g and 5 min at 3000 × g), variation was calculated as the coefficient of variation (CV) using the formula

$\mathrm{CV}(\%)=100\times \frac{s}{x}$

where $s=\sqrt{\frac{\sum (x2-x1)2}{2N}}$ , x = mean, N = number, x¹ = result from tube 1, x² = result from tube 2. The calculated CVs thus represent not only analytical variation but also variation from centrifugation and tubes. Calculated CVs were compared with CV goals, which refer to limits for maximum allowable variation used in laboratory routine quality control. The calculated CVs were also compared with state of the art analytical CV for the analyte, found as the average monthly analytical variation in laboratory routine quality control data during the investigation period (September–December 2014).

Laboratory routine quality control goals for bias and CV were determined from within- and between-subject biological variation or empirically (CA and FT3).^12,13

Results

Results are summarized in Table 1. The mean differences between measurements obtained after centrifugation for 5 min at 3000 × g compared with 10 min at 2200 × g were small (≤1.2%) and well within the acceptance limit (bias goal) for all analytes except LDH.

Table 1.

Mean difference and CV for selected analytes measured in samples centrifuged for 5 min at 3000 × g compared with 10 min at 2200 × g.

Analyte	Unit	Material	n	Median (range)	Local reference interval (adults)	Bias (%)		CV (%)
						Mean difference (95% CI)	Goal	10 min 2200 × g	5 min 3000 × g	Goal	Analytical
B12	pmol/L	P	40	315 (177–1407)	200–600	0.2 [−1.0–1.4]	17.7	4.9	4.2	11.3	7.9
		S	40	288 (117–892)		0.3 [−0.8–1.4]		5.0	4.2
Ca	mmol/L	P	40	2.26 (2.02–2.44)	2.20–2.55	−0.1 [−0.4–0.3]	2.0	1.2	1.0	2.5	1.0
		S	40	2.26 (1.85–2.91)		0.1 [−0.2–0.3]		0.6	0.7
Folate	nmol/L	P	39	12.7 (4.4–43.6)	>6.0	−0.2 [−1.4–1.0]	19.2	2.2	4.4	12.0	4.1
		S	40	12.2 (5.5–37.9)		0.5 [−1.1–2.0]		2.7	3.5
FT3	pmol/L	P	40	3.9 (1.6–6.4)	3.9–6.8	1.2 [−0.8–3.2]	7.2	3.7	4.4	7.5	4.3
		S	30	3.9 (1.7–5.6)		0.6 [−0.8–2.1]		4.3	3.4
IgM	g/L	P	40	0.77 (0.24–1.95)	0.39–2.30	−0.1 [−0.6–0.4]	11.9	1.4	1.5	3.0	1.4
		S	39	0.64 (0.06–3.23)		0.7 [0.0–1.4]		1.3	1.4
K	mmol/L	P	40	3.8 (2.8–5.2)	3.5–4.6	0.3 [−0.1–0.7]	1.8	1.0	1.5	2.4	0.8
		S	40	4.1 (3.4–4.7)		0.2 [−0.2–0.7]		1.7	0.8
LD	U/L	P	40	177 (83–482)	105–255	6.3 [4.1–8.5]	4.3	3.3	5.0	4.3	2.6
P	mmol/L	P	40	1.08 (0.66–1.78)	0.71–1.53	−0.2 [–0.6–0.2]	3.2	1.3	1.5	4.3	1.4
		S	40	1.10 (0.57–1.40)		0.2 [−0.3–0.8]		1.5	1.3
TP	g/L	S	40	66 (52–76)	62–78	0.3 [−0.2–0.8]	1.8	1.0	1.1	2.0	0.6

P: plasma, S: serum.

The individual differences between sample pairs were assessed using difference plots shown in Figure 2 for plasma samples and Figure 3 for serum samples. The difference plot for LDH revealed several differences exceeding the total error acceptance limit, and overall, the results were higher after centrifugation for 5 min at 3000 × g compared with the standard protocol of 10 min at 2200 × g. For TP (Figure 3) and plasma FT3 (Figure 2), two results in the difference plot stood out from the remaining results and exceeded the total error acceptance limit. There was nothing unusual about the samples in question based on visual inspection, and the HEM, ICT and LIP index measurements indicated no haemolysis, icterus or lipaemia. Otherwise difference plots for all analytes except LDH showed no systematic or concentration-dependent differences and individual differences were within acceptance limits.

Figure 2.

Difference plots for plasma. X-axis: mean of all four samples (1 P + 2 P + 3 P + 4 P / 4). Y-axis: ([mean 5 min 3000 × g−mean 10 min 2200 × g]/mean 10 min 2200 × g) × 100%. Solid thin horizontal line equals line of identity. Solid bold lines equals total error (TE = [1.65 × CV goal] + bias goal). Vertical dotted lines depict reference range for adults. The HEM graph included an additional data point (0.18, 0.38) not shown in this figure.

Figure 3.

Difference plots for serum. X-axis: mean of all four samples (1 S + 2 S + 3 S + 4 S / 4). Y-axis: ([mean 5 min 3000 × g−mean 10 min 2200 × g]/mean 10 min 2200 × g) × 100%. Solid thin horizontal line equals line of identity. Solid bold lines equals total error (TE = [1.65 × CV goal] + bias goal). Vertical dotted lines depict reference range for adults.

Total CV was determined for results in each of the two centrifugation settings, considering measurements as double determinations even though they were actually made as single measurements on two different tubes from the same venepuncture. This CV thus included not only analytical variation but also tube- and centrifugation variation. Hence, if the calculated total CV met the CV goal, then variation from centrifugation alone would also be expected to meet the criteria. As can be seen in Table 1, the CV for LDH was higher than acceptable (CV goal) for samples centrifuged for 5 min at 3000 × g, while the CV was within the acceptance criteria (≤CV goal) for samples centrifuged according to laboratory routine practice (10 min at 2200 × g). For all other analytes, the total CV was within the acceptable limit (≤CV goal). Table 1 also states the analytical CV for each analyte in order to estimate how much of the total variation can be accounted for by analytical variation.

For serum IgM and plasma FOL, the results from one sample set (four tubes) were outside the measuring range and were excluded from the data evaluation.

Visual inspection of all tubes following centrifugation did not reveal any differences between the two centrifugation settings regarding incidence or frequency of visible haemolysis and quality of plasma/serum separation from blood cells. HEM index, ICT index and LIP index measurements were generally low in all tubes for both centrifuge settings. Two plasma tubes centrifuged for 5 min at 3000 × g and one serum tube centrifuged for 10 min at 2200 × g had an HEM index greater than allowed for LDH and FOL in laboratory routine samples, and hence the results on these tubes were excluded from the data evaluation for LDH and FOL. Apart from these three tubes, the HEM index measured was ≤0.06 g/L in all plasma tubes and ≤0.17 g/L in all serum tubes, regardless of the centrifugation protocol used. In all plasma tubes, the measured ICT index was ≤24.8 µmol/L and the LIP index was ≤0.18 mmol/L, and in all serum tubes, the ICT index was ≤25.3 µmol/L and the LIP index was ≤0.10 mmol/L. Difference plots for HEM index, ICT index and LIP index are shown in Figures 2 and 3. Interference index measurements on the Architect c16000 are determined mathematically based on spectrophotometric reads of plasma or serum diluted with isotonic saline, and concentration units are only approximate.

Discussion

Centrifugation settings can be altered to optimize sample flow and turnaround time in the clinical laboratory, and it is important to ensure that the altered centrifugation settings do not compromise analytical quality. We compared an accelerated centrifugation time of 5 min at 3000 × g to a standard protocol of 10 min at 2200 × g using BD Vacutainer serum and plasma collection tubes without gel separator. Manufacturers typically recommend centrifugation for 10 min or more for lithium-heparin plasma and serum tubes, and some recommend higher g-force for tubes with gel separator in order to provide a stable and well-defined gel layer.^1,2 A few previous studies have investigated centrifugation times shorter than 10 min using different serum or plasma collection tubes, g-forces and analytical methods, and in general they have found evidence that centrifugation time can be decreased to 4–7 min without adversely affecting the analytical quality.^6–9 Apart from the study of Koenders et al., ⁸ these studies were all performed using collection tubes with gel separator. Although gel separator tubes are widely used and may be advantageous in that they reduce the need to aliquot specimens, there are also disadvantages. Separator gel flotation can occur in specimens with a high protein content, and this can lead to mechanical disruption upon aspiration of the gel into instrument sampling probes. ¹⁰ Furthermore, gel separators have been shown to affect measurements of parameters such as therapeutic drugs,^14,15 androstendione ¹⁶ and metabolomics ¹⁷ due to absorption or interaction. In our study, using collection tubes without gel separator, confirmation has been found that a centrifugation of 5 min at 3000 × g can be used for chemistry and immunochemistry analytes. However, for LDH, we found an increased coefficient of variation (CV = 5.0%) and higher results (mean bias = 6.3%) after centrifugation for 5 min at 3000 × g. This finding is in contrast to previous studies, where LDH was not adversely affected at centrifugation times of 4–7 min.^6–9 Three of these studies all used a lower g-force of around 1900 × g compared with 3000 × g in this study.^7–9 A potential concern with centrifugation at higher g-force is in vitro lysis of blood cells, due to increased mechanical stress. LDH is present in large amounts in red blood cells and platelets and any lysis would lead to overestimation of it.^18–20 Two plasma tubes centrifuged for 5 min at 3000 × g had overt haemolysis with HEM index > 0.25 g/L. The tubes were from two different sample pairs (see Figure 1), and both of the corresponding tubes in each pair had no evidence of haemolysis (HEM index values were 0 g/L). Since only one tube of a sample pair was haemolysed, it is unlikely that the haemolysis was caused by the more rapid centrifugation. For the remaining 38 plasma tubes, the HEM index measured on Abbott Architect was very low (≤0.06 g/L) indicating a very low degree of haemolysis of red blood cells even with the accelerated centrifugation of 5 min at 3000 × g. This finding is supported by Mensel et al., ⁶ who investigated BD Vacutainer serum SST II Advance tubes with gel separator specially designed for shorter centrifugation time, using centrifugation for 5 min at 3000 × g and found that this centrifugation mode did not increase the rate of haemolysis. In addition to haemolysis of red blood cells, the lysis of platelets is also a potential source of LDH. The IFCC recommendation for LDH measurement ²¹ states serum as the preferred sample material for LDH in order to avoid the presence of platelets, but in daily practice lithium-heparin plasma is often preferred, because it is more rapidly available, hence the use of plasma for LDH in this study. According to CLSI guideline H21-A5, ²² a platelet-poor plasma can be obtained by centrifuging at 1500 × g for at least 15 min, corresponding to a total of 22,500 g/min. It is therefore possible that using the accelerated centrifugation setting (5 min at 3000 × g equal to 15,000 g/min) will lead to more platelets remaining in the plasma than there are using the standard setting (10 min at 2200 × g equal to 22,000 g/min) and that this will result in an increase in LDH activity. It is of interest that we did not find any increased plasma values for K and P, analytes that are also released from platelets. Whether the observed increase in LDH is in fact due to lysis of platelets or an optical interference caused by intact platelets ²³ remains to be investigated.

In conclusion, under the specified conditions described, we found that a centrifugation protocol of 5 min at 3000 × g can be used for all of the general chemistry and immunochemistry analytes studied, without compromising the quality of the analytical results, with the exception of LDH. For LDH, we found that centrifugation for 5 min at 3000 × g increased the results obtained, with a mean bias of 6.3% and a higher CV of 5% compared with the standard protocol of 10 min at 2200 × g. The clinical impact of these changes must be assessed in relation to ongoing demands for faster turnaround times and analytical results, which can in part be met by shorter centrifugation times.