Stability of chemical and immunochemical analytes in uncentrifuged plasma samples

Abstract

Background

The stability of analyte concentrations in plasma after prolonged contact with blood cells in uncentrifuged lithium-heparin gel tubes was studied.

Methods

To investigate the stability of concentrations of 26 chemistry and 15 immunochemistry analytes, the simultaneously drawn samples (n = 50) were measured after 6 h storage at +8°C and +22°C in whole blood and after immediate separation of plasma. The analyte concentrations were measured with a Roche Modular PPEE analyser using reagents from Roche Diagnostics.

Results

After prolonged contact with cells a clinically significant change was only observed for potassium where the mean value increased from 4.0 mmol/L to 4.8 mmol/L (P < 0.001) when stored at +8°C.

Conclusion

Immediate separation of plasma from cells is recommended. However, when prolonged contact of plasma with cell is unavoidable, samples can be kept uncentrifuged for up to 6 h at +8°C or at +22°C. The stability of potassium, however, is temperature-dependent and cannot be measured from refrigerated blood samples.

Introduction

Plasma-based testing has increased in many laboratories to reduce turnaround time by shortening the time interval between blood collection and testing. However, a common problem in the daily routine facing clinical laboratories is the uncertainty of the integrity of uncentrifuged specimens for chemical and immunochemical analyses. It is very well known that, ideally, the plasma should be separated from cells as quickly as possible to prevent the ongoing metabolism of cellular constituents. ¹ However, the allowance of patient specimens to prolonged contact with blood cells would offer more flexibility to unload the early morning rush hours in many laboratories and loosen the requirements of transportation of specimens to testing laboratories. Publications on analyte stability in uncentrifuged heparin plasma specimens are few and concern mainly the stability of the concentrations of chemical analytes. ^1–4

In this study we determined the stability of concentrations of 26 chemical and 15 immunochemical analytes after 6 h of storage in whole blood. In addition, the influence of storage temperature and transportation (courier or bus) of the tubes on analytical stability was examined.

Materials and methods

Venous blood was simultaneously collected from 50 fasting volunteers into three 3.5 mL VenoSafe™ lithium heparin gel tubes (Terumo Europe, Belgium). Cell-free plasma was obtained after 6 h storage at +22°C and +8°C in whole blood and after immediate (within 0.5 h) separation of plasma. All plasma samples were centrifuged at 2500 g for 10 min at +18°C according to manufacturer's instructions (Terumo Europe, Belgium).

All analytes were measured from a single tube with one Roche Modular PPEE analyser, with commercial reagents provided by Roche Diagnostica (Roche Diagnostics, Germany). Thus, components such as glucose, demanding a special blood sampling tube for a longer stability were not considered.

Statistically significant changes were determined for each analyte by one-way analysis of variance (ANOVA) or Kruskal-Wallis test. Statistical analyses were performed using SPSS 16.0 (SPSS Inc, Chicago, IL, USA).

To determine the changes in analyte concentrations of plasma with and without prolonged contact with cells, the mean from 50 volunteers for each respective analyte was obtained. Clinically significant changes were determined by the significant change limit ⁵ (SCL) approach defined as:

SCL = Initial value ± 2.8 usual SD.

It is based on the assumption that the usual SD (USD) is representative of the inherent day-to-day variability of the method. In this study, the calculated mean for each analyte at 0.5 h represented the initial value. USD was obtained by averaging the SD of the quality-control data (target mean most closely matched the 0.5 h mean) observed for the last 12 months for each respective analyte. ⁵ SCL was calculated by establishing the range (±2.8 USD) from mean at 0.5 h.

Results and discussion

Table 1 shows the statistics for plasma analyte concentrations obtained in samples separated immediately after collection, along with those of 6 h storage as whole blood. The results indicate that the concentrations of 25 chemical and 15 immunochemical analytes were quite stable when samples were stored uncentrifuged for 6 h either at +22°C or +8°C. Only for potassium did the observed mean exceeded the SCL, increasing from 4.0 mmol/L to 4.8 mmol/L (P < 0.001) when stored at +8°C. This is known to be due to the Na⁺, K⁺-ATPase activity brought about by cold. ⁶ Our results are in accordance with the recently published study of Tanner et al. ⁷ although their findings are based on serum.

Table 1

Stability of plasma analytes after 6 h storage in whole blood

Analyte (units)	Range*	Storage time and temperature			USD value	SCL range‡
		0.5 h at +22°C, mean†	6 h at +22°C, mean†	6 h at +8°C, mean†
P–ALP (U/L)	14–1202	114	113	114	4	100–124
P–ALB (g/L)	16–45	33	33	33	0.77	32–36
P–ALT (U/L)	8–393	48	50	49	2	41–53
P–AMYL (U/L)	5–172	53	53	53	3	44–62
P–BIL (μmol/L)	4–350	27	27	27	1	23–29
fP–Ca (mmol/L)	1.84–2.47	2.22	2.23	2.20	0.040	2.10–2.34
P–CK (U/L)	5–1042	118	120	117	5	102–132
P–Cl (mmol/L)	90–110	104	103	103	2	98–110
P–CRP (mg/L)	0–305	43	44	43	2	35–47
P–Fe (μmol/L)	2–35	10.8	11.6	11.1	0.5	9.5–12.5
P–GGT (U/L)	6–1075	100	100	100	3	87–105
P–HAPTO (g/L)	0–4.6	2.0	2.0	2.0	0.08	1.7–2.1
P–K (mmol/L)	3.0–5.6	4.0	4.0	4.8§**	0.08	3.7–4.1
P–CHOL (mmol/L)	1.9–9.7	4.1	4.1	4.0	0.15	3.7–4.6
P–CHOL–HDL (mmol/L)	0.21–3.10	1.14	1.14	1.10	0.040	1.02–1.26
fP–CREA (μmol/L)	46–194	83	84	84	4	71–95
fP–LDH (U/L)	101–831	264	276	282	9	237–291
P–Mg (mmol/L)	0.65–1.12	0.95	1.01§	0.97	0.050	0.80–1.10
P–Na (mmol/L)	127–146	139	140	138	1.5	135–144
fP–PHOS (mmol/L)	0.67–1.59	1.09	0.96§	1.12	0.05	0.95–1.25
P–RF (IU/mL)	0–121	4	4	4	1.0	4–10
P–Transferrin (g/L)	0.9–3.2	2.1	2.1	2.1	0.12	1.9–2.6
P–TfR (mg/L)	0.7–5.7	2.0	2.0	2.0	0.15	1.6–2.5
fP–TG (mmol/L)	0.53–5.21	1.47	1.47	1.48	0.1	1.17–1.77
P–UA (μmol/L)	117–861	359	355	361	7	338–380
P–BUN (mmol/L)	1.7–19.3	6.3	6.2	6.2	0.25	5.5–7.0
P–B12 (pmol/L)	149–1476	514	490	510	30	416–596
P–CA12–5 (kU/L)	3–818	57	57	57	2.7	49–65
P–CA15–3 (kU/L)	6–44	16	16	16	1.3	12–20
P–CA19–9 (kU/L)	1–350	22	22	22	1.2	18–26
P–CEA (μg/L)	0.2–5.7	2.1	2.2	2.1	0.30	1.2–3.0
P–CK–MBm (μg/L)	1.1–90.7	5.2	5.2	5.2	0.23	4.4–5.8
fP–C–PEPT (nmol/L)	0.3–4.1	1.2	1.2	1.2	0.08	0.1–1.4
fP–INSU (mU/L)	1–478	28	26	27	1.3	23–31
P–CORSOL (nmol/L)	181–1750	503	504	512	15	444–534
fP–PTH (ng/L)	24–187	61	57	60	3.8	50–72
P–PSA (μg/L)	0–21.7	2.7	2.7	2.7	0.18	2.3–3.3
P–FPSA (μg/L)	0–2.0	0.40	0.38	0.39	0.02	0.34–0.46
P–TnT (μg/L)	0.01–4.22	0.36	0.36	0.36	0.080	0.10–0.58
P–TSH (mU/L)	0.50–13.03	2.15	2.15	2.15	0.270	1.54–3.16
P–FT4 (pmol/L)	10.1–24.3	16.0	16.5	16.4	1.24	12.2–19.6

SCL, significant change limit; USD, usual standard deviation; ALP, alkaline phosphetase; ALB, albumin; ALp, alanine transaminase; AMYL, amylase; BIL, billrubin; Ca, calcium; CK, creatinine kinase; CI, chloride; CRP, c-reactive protein; Fe, iron; GGT, gamma glutamyl transferase; HAPTO, haptoglobin; K, potassium; CHOL, cholesterol; CREA, creatinine; LDH, lactate hydrogenase; Mg. magnesium; Na, sodium; PHOS, inorganic phosphate; RF, rheumatoid factor; TRANSF, transferrin; TfR, transferrin receptor; TG, triglycerides; UA, uric acid; BUN, urea; B12, vitamin B12; CEA, carcino embryonal antigen; C-PEPT, C-peptide; INSU, insulin; CORSOL, cortisol; PTH, parathormone; PSA, prostate specific antigen; FPSA, free PSA; TnT, troponin T; TSH, thyroid-stimulating hormone; FT4, free T₄

*Range of results from 0.5 h concentration at +22°C (n = 50)

^†Means of results (n = 50)

^‡Mean at 0.5 h±2.8 USD

^§Statistically significant difference from 0.5 h concentration (one-way analysis of variance or Kruskal-Wallis test, P < 0.05)

**SCLs have been exceeded

In order to study the effect of transportation (courier or bus) on the stability of analyte concentrations, the conditions were simulated by strong-handed manipulation of the tubes. Neither the continuous gentle swinging in a mixer nor substantial upside down turning of the uncentrifuged tubes during the prolonged storage had an additional effect on measured concentrations of analytes.

Our results indicate that in the case when lithium-heparin plasma specimens cannot be separated after collection, the storage either at +22°C or +8°C up to the 6 h can be allowed since most concentrations of analytes were sufficiently stable when compared with those immediately separated from cells. The stability of potassium, however, is temperature-dependent and cannot be measured from refridgerated blood specimen.