Sodium Fluoride tubes versus plain tubes for In vitro blood glucose analysis

containing tubes were introduced into laboratory practice for blood collection in determination of glucose[3]. It is known that NaF shows its antiglycolytic effect by inhibiting the enolase enzyme of the glycolytic pathway in erythrocytes[4]. However, it has been recognized that the action of NaF towards enolase is slow as it will start at least in four hours from the time of collection of blood[4,5]. Hewage N I et al


Background
Diabetes Mellitus (DM) is a common metabolic derangement debilitating populations in both characterized by high blood glucose levels over a prolonged period [1]. Blood glucose analysis is vital for both the diagnosis and monitoring of therapy in DM are narrow and therefore there is an increased need for reliable results to classify individuals accurately [2].
In the routine laboratory set-up blood specimens for glucose determination are being collected into tubes containing tubes were introduced into laboratory practice for blood collection in determination of glucose [3]. It is known that NaF shows its antiglycolytic effect by inhibiting the enolase enzyme of the glycolytic pathway in erythrocytes [4]. However, it has been recognized that the action of NaF towards enolase is slow as it will start at least in four hours from the time of collection of blood [4,5].
al., reported that there was no difference in blood and NaF tubes which were separated within two of NaF tubes has been reviewed by the American Diabetic Association (ADA) laboratory guidelines for the diagnosis and management of DM with its recommendation to stop use of NaF as the only antiglycolytic agent [2].
It has been further pointed out that the tubes containing NaF are useful if there is a delay in the separation of plasma from cellular components for several hours [5,6]. Studies done by Chan et al., however, showed that following four hours, the concentration of glucose in whole blood in the two hours at room temperature and recommended the use of NaF if there is a delay in separating [7]. Considering the slow activity of NaF towards enolase enzyme, the use of NaF tubes to collect blood for this core biochemical test has to be revisited [4,6].
In turn, the specimens collected into the tubes containing NaF are not suitable for the measurement of other key anylates such as Na+, K+ and enzymes, and therefore multiple different collection containers are required.

Objectives
values obtained using plasma NaF/K 2 C 2 O 4 tubes and serum plain tubes in a routine laboratory setup, and to assess the changes in glucose levels in plasma and serum up to six hours from the separation of cellular components. Collection of blood into plain tubes is more convenient especially when it is collected from critical care patients where multiple blood samples are needed. On the contrary, the use of the historical tubes (NaF as the preservative) is not revised though there is a great advancement in the technology today.

Materials and methods
Subjects of the Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka, during a period of six months (from March to September 2017). Sixty one participants (healthy volunteers) was ethically approved by the Ethics Review Committee, Faculty of Medical Sciences, University of Sri Jayewardenepura (Protocol approval No. MLS/08/2017).

Methods
During the study period, blood samples (2.5 ml) were collected into both plain and NaF/K 2 C 2 O 4 tubes. Following centrifugation of plasma (NaF/ K 2 C 2 O 4 ) and serum (plain tube), the centrifuged tube sets were allowed to stand for 1, 2, 4 and 6 hours. Glucose concentrations were measured in both plasma and serum by glucose oxidase method (DiaSysGOD FS reagent kit) using Shimadzu UV 1601 visible spectrophotometer (Simadzu Corporation, Kyoto, Japan, % CV, 0.81) [8]. For both plasma (in NaF/K 2 C 2 O 4 tubes) and serum (in were made based on the serum separation time. At each point of analysis the serum or plasma was in contact with the blood cells in the corresponding values and glucose concentration at one, two, four and six hours after serum or plasma separation. Samples which did not show haemolysis were included in the comparison. Each day before sample processing, internal quality control was performed using two reference glucose solutions with higher and lower glucose were 290.9 ± 10 mg/dl (high) and 94.4 ± 10 mg/dl (low) respectively. Meanwhile, the control glucose solution was also run in each day to ensure the reproducibility of the spectrophotometer. All the reagents were kept inside the refrigerator at 0ºC to glucose samples were aliquoted into eppendorf tubes and they were kept at -20ºC.

Results
Glucose concentrations observed in plasma (NaF/ K 2 C 2 O 4 ) and serum (plain tubes) up to six hours post separation and the percentage reduction in comparison with baseline value are depicted in 2 C 2 O 4 ) and serum (plain tube) glucose values (n=61, paired to two hours of separation (P>.05). In fact, blood specimens collected into plain tubes are much better since they had shown minimal reduction up to two hours post separation. Nevertheless, glucose concentrations measured after four and six hours in

and plain tubes
Discussion should be used instead of NaF/K 2 C 2 O 4 tubes to collect blood for glucose measurements with accepted values under routine laboratory setup. In both types of tubes, clinically equivalent results were yielded given that the blood sample processing had an optimum time of less than two hours from collection to the time of analysis. of previous investigators [3,5]. With the progress and improvements of the technology, it has been revealed that serum analyzed for glucose within reasonable time after the collection (less than two hours) gives same blood glucose values as in plasma serum/ plasma documented in American Diabetes Association (ADA) guidelines is thirty minutes glucose values collected to NaF/K 2 C 2 O 4 has been observed by several researchers and the difference reported was proportional to the time delay [5,9,10].
suitable for blood collection if there is a long delay in the separation of plasma from cells [10]. According to Chan et al., NaF has a minimal preservative effect within two hours of blood collection [7]. Its effectiveness was reported after four hours of blood collection and activity persisted at least for three days at room temperature.
Use of plain tubes for glucose measurements, offer many specimen processing advantages; these tubes can be used for the majority of biochemistry and immunology tests thus reducing the need for other consumables (additives) and the amount of blood drawn from the patients. If NaF/K 2 C 2 O 4 tubes are replaced with plain tubes, several laboratory tests including glucose can be performed from a and geriatric patients where venipuncture may be in state sector hospitals in countries such as Sri Lanka where free health care is offered.
Further, it was observed that, the blood samples collected in NaF/K 2 C 2 O 4 tubes had the higher rate of haemolysis (11%) when compared with that in plain tubes (4%). Most previous studies have shown similar results [3,5]. Presence of larger crystals of NaF/K 2 C 2 O 4 could rupture the RBC resulting in the higher rate of haemolysis [11]. Release of glucose from RBC has a positive bias and release of catalase has a negative bias on actual glucose value, thus raising a controversy in validity of results [12].

Conclusion
acceptable blood glucose values could be obtained by using plain tubes instead of NaF/K 2 C 2 O 0 tubes if the analysis is performed within a reasonable time of less than two hours following blood collection. However, if analysis of blood glucose is delayed or expected to be delayed for more than two hours tubes could be acclaimed for glucose measurement.

Disclosures
funding was received for this work.