A Stabilizing Agent, PCA/DTPA, Improves Plasma Storage Life for the Chromsystems Vitamin C Assay up to Six Months
2021; 41(4): 414-418
Ann Lab Med 2020; 40(6): 448-456
Published online November 1, 2020 https://doi.org/10.3343/alm.2020.40.6.448
Copyright © Korean Society for Laboratory Medicine.
Laboratori Clínic Territorial Metropolitana Sud–Hospital Universitari de Bellvitge. Hospitalet de Llobregat, Barcelona, Spain
Correspondence to: Ariadna Arbiol-Roca, Ph.D.
Laboratori Clínic Territorial Metropolitana Sud–Hospital Universitari de Bellvitge, 08907 Hospitalet de Llobregat, Barcelona, Spain
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
The storage temperature and time of blood gas samples collected in syringes constitute preanalytical variables that could affect blood gas or lactate concentration measurement results. We analyzed the effect of storage temperature and time delay on arterial or venous blood gas stability related to pH, partial pressure of carbon dioxide (
In total, 1,200 arterial and venous blood sample syringes were analyzed within 10 minutes of collection. The samples were divided into different groups to determine parameter stability at 25, 4–8, and 0–3.9°C and at different storage times, 60, 45, 30, and 15 minutes. Independent sample groups were used for each analysis. Percentage deviations were calculated and compared with acceptance stability limits (1.65× coefficient of variation). Additionally, sample group sub analysis was performed to determine whether stability was concentration-dependent for each parameter.
The pH was stable over all storage times at 4–8 and 0–3.9°C and up to 30 minutes at 25°C.
The strictest storage temperature and time criteria (0–3.9°C, 45 minutes) should be adopted for measuring pH,
Keywords: Stability, Blood gases, Storage, Temperature, Time, Preanalytical variables.
Most errors affecting total blood analysis occur during the preanalytical phase, mainly owing to a lack of standardized procedures for samples collection, for example, the correct homogenization of samples and removal of air bubbles . The storage temperature and time of the syringes with collected samples are additional preanalytical variables that can affect results of blood gases or lactate concentration . To minimize the influence of these preanalytical variables on the results, the laboratory are responsible to inform practitioners about the preanalytical conditions under which samples should be collected, stored, and transported .
Some guidelines recommend analyzing blood samples within 30 minutes after collection and keeping them at a low temperature [4–7]; however, there is no agreement between the biochemical parameters and sampling conditions for syringes with collected samples in the literature. Previous studies have used different criteria for their calculations based on statistical, metrological, or biological variability, and as a result, suggested different recommendations [8–13]. Therefore, establishment of stability limits depends on the mathematical criterion defined. Laboratories may calculate their own stability limits under specific sample storage conditions using the mathematical criteria recommended by scientific societies or clinical guidelines [14, 15].
Previous studies have investigated stability effects [12, 13, 16], kind of syringes [8, 13, 17], different storage temperatures [7–9, 13, 18], transport conditions , and even syringe volumes [12, 13]. However, despite the apparently vast bibliography, there is poor consensus about blood gas stability in daily practice. Therefore, we performed the main stability study with a large number of samples. We aimed to analyze the effects of temperature and duration of storage on the arterial or venous blood gas stability in samples in terms of pH, partial pressure of carbon dioxide (
The study, performed in the Stat Laboratory of Bellvitge University Hospital, Hospitalet de Llobregat, Barcelona, Spain, was approved by the Clinical Research Ethics Committee of Bellvitge University Hospital (Ref. PR297/12).
The study was performed with blood samples obtained from the Intensive Care Unit and the Emergency Department. From June 2012 to January 2013, 1,200 arterial and venous blood samples were collected into SafePICO (Ref. 956–622; Radiometer) and Marquest’s Quick ABG syringes (Ref. 4023TRU; Vyaire Medical, Höchberg, Germany); both syringes contain dried lyophilized lithium heparin (60–100 UI) as an anticoagulant. The study team collected the samples at the patient’s point-of-care to ensure that these reached the laboratory in less than 10 minutes after collection. Informed consent was not obtained from the patients because the samples were randomized and anonymized. We collected a sufficient number of samples to ensure a minimum of 30 samples for each parameter at each specific time. Following the elimination of aberrant data pairs, a total of 1,147 samples were used.
The blood sample syringes used in our study were considered residual material; therefore, instead of discarding the samples once analyzed and validated, they were incubated at specific goal temperatures. In fact, each syringe was analyzed twice (time 0 minute and then incubated at the goal temperature for 60, 45, 30, or 15 minutes, respectively).
Before analysis, all samples were thoroughly mixed for 5–10 seconds by vertical hand-rolling, and any visible air bubbles were carefully removed. The pH,
Following the first analysis, the samples were grouped into three categories according to the storage temperature over 60 minutes: the first group samples were stored at 25°C, the second group samples at 4–8°C, and the third group samples at 0–3.9°C in an ice-water bath. Samples were randomly left at each goal temperature. The temperature was continuously monitored using a mercury-in-glass thermometer.
The stability study began by storing the samples for 60 minutes. After the desired time at the indicated temperature, the samples were quickly hand-mixed and reanalyzed. The first and second results were stored in a database, and statistical calculations were performed. If any parameter was not stable ≤60 minutes, the study of that sample group started again with new samples, for 45 minutes. If stability was still an issue, the experiment was carried out for 30 and 15 minutes, sequentially. Independent sample groups were used each time (Fig. 1).
For the main study, stability was assessed with all data. For the substudy, all data were sorted into groups based on the initial values; pH,
Acceptance stability limits (S) were obtained based on the Sociedad Española de Medicina de Laboratorio criteria for maximum allowable bias (S=±1.65 coefficient of variation [CV]) . The within-run imprecision, expressed as CV, was calculated from the differences between pairs of duplicate measurements analyzing at least 60 samples using the Dahlberg formula (s=√ ∑
The percentage deviation (PD) between the first measurement (
Statistical calculations were performed with Microsoft Excel 2010 (Microsoft Corp., Redmond, WA, USA), and the Bland-Altman method was performed using Analyse-IT software (Analyse-IT software Ltd., Leeds, UK). The level of significance was defined at
The CV values of the main study (overall data) and the substudy (by concentration group) are shown in Table 1. All CV values achieved the maximum allowed values according to the metrological requirements of our laboratory. S was calculated with these CV values.
Blood pH was stable over all time periods when the sample was preserved via refrigeration (0–3.9°C or 4–8°C) or up to 30 minutes at 25°C. For
Table 3 shows the results of the samples sorted according to the initial value of the blood gas parameters.
Blood pH was stable for all storage times when it was preserved at 4–8 or 0–3.9°C, for the three groups. It was also stable for 30 minutes at 25°C in the group below the lower limit of the reference interval and for 15 minutes within and above the upper limit of the reference interval. Similarly,
Previous stability studies regarding the measurement of blood gases are outdated and present contradictory data. Each study differs in terms of design, blood sample volume, initial oxygen concentration, syringe type, and size, resulting in different conclusions [8–13]. We carried out a stability study with added value using a large number of samples and by grouping the samples by the initial value of each parameter, to determine whether stability is concentration-dependent. Our findings agree with the findings by Srisan,
Cell metabolism at 25°C could explain this phenomenon, as CO2 is generated both aerobically and anaerobically, leading to an increase in
Considering our results, we recommend cooling (0–3.9°C) sample syringes when
The sample extraction procedure should be rigorous since air aspiration or bubble formation in blood gas syringes can significantly alter blood gas parameters . Exposure of a blood gas sample to air would typically result in an elevation or drop in
Finally, lactate is a degradation product of the anaerobic pathway and may contribute to the decrease in pH. When blood sample is collected, metabolism in blood cells is still active; blood cells consume oxygen and glucose, the sample becomes anaerobic, and lactate is produced, resulting in concomitant acidosis .
Our study has some limitations. First, the stability time was calculated not from sample collection but from the reception of syringe at the laboratory, taking into account that not more than 10 minutes pass between collection and analysis. We cannot exclude some degree of gas exchange in syringes that were opened during analysis, even though we re-capped them at the earliest. The second limitation concerns the samples, as we performed the study with arterial and venous samples in order to cover all measurement ranges. To calculate the PD, we mixed the arterial and venous blood gas results. The correlation between arterial and venous blood results is good for pH,
In summary, requests for blood pH,
Study flow diagram. The stability study began with storing the samples for 60 minutes. If any parameter was not stable for ≤60 minutes, the study was started again with new samples for 45 minutes. If stability was still an issue, the experiment was carried out for 30 and 15 minutes, sequentially. Independent sample groups were used each time. Measurements were conducted using an ABL800 analyzer (Radiometer, Copenhagen, Denmark).
Mean bias from baseline for pH (A),
Within-run imprecision in overall group and in groups sorted by concentration
|Within-run imprecision||Mean (N) overall|
|Mean (N) by groups|
|CV requirement (%)||CV overall (%)|
|CV by groups (%)|
|pH||<7.35||7.39 (90)||7.30 (30)||0.20||0.06||0.07|
|<35.0||45.4 (90)||30.7 (30)||4.00||2.02||1.68|
|> 52.0||64.0 (30)||2.07|
|<83.0||108.6 (91)||45.9 (31)||7.00||5.62||5.35|
|≤ 95.0||83.1 (61)||68.9 (31)||7.00||2.27||3.88|
|Lactate (mmol/L)||≤ 1.84||2.35 (60)||1.20 (30)||13.00||3.57||5.75|
Evaluation of sample storage temperature (25, 4–8, and 0–3.9°C) and duration of biological parameter stability (in minutes) in all samples (overall group–main study)
|N||Temperature (°C)||Time (min)||PD (95% CI) (%)*||S (%)†|
|84||25||<15||10.2 (7.25; 13.1)||9.27|
|190||4–8||<15||12.2 (10.7; 13.6)|
|104||4–8||<15||7.68 (6.59; 8.76)|
|Lactate||65||25||<15||14.0 (10.8; 17.1)||5.89|
|39||4–8||<15||8.13 (6.46; 9.81)|
Evaluation of sample storage temperature (25, 4–8, and 0–3.9°C) and duration of biological parameter stability (in minutes) sorted by the initial concentration value (substudy)
|Temperature (°C)||N||Time (min)||PD (%)*||S (%)†||N||Time (min)||PD (%)*||S (%)†||N||Time (min)||PD (%)*||S (%)†|
|Lactate≤1.84 mmol/L||Lactate>1.84 mmol/L|