To ensure its function of transporting hemoglobin and maintaining its existence, the red blood cell must constantly fight against two dangers; oxidation of its constituents; iron, hemoglobin and membrane proteins and its own hyperhydration. The means of this fight are provided by intra-erythrocyte glycolysis. This physiology explains the high consumption of glucose by red blood cells which is why blood is taken from a glycolysis-inhibiting anticoagulant when plasma glucose is to be determined  . Diabetes screening and diagnosis, a major public health problem are based on threshold values for blood glucose. The determination of blood glucose is thus one of the most frequently performed analyses in hospitals as in city laboratories. If the analytical phase is perfectly controlled, some steps of the pre-analysis phase, especially the transmission delay, are less standardized and therefore condition the quality of the results. There is evidence of a lack of serum glucose stability or serum plasma stability   . They are mostly old and have little evidence of stability in whole blood with or without antiglycolics. However, many laboratories are receiving non-centrifuged specimens from remote collection sites with varying delivery times, sometimes several hours. To overcome this problem, manufacturers of tubes for the collection of biological samples have proposed for several years the use of antiglycolytic agents, which are supposed to guarantee the stability of this parameter over time  . The most frequently used antiglycolytics are sodium fluoride and monoiodoacetate but unfortunately sodium fluoride creates hemolysis and the anti-glycolytic action of mono-iodoacetate is noticeable beyond two hours  . As part of the laboratory quality approach, and in particular the reflection conducted on the preanalytical step, we were led to study the instability of glycemia on lithium heparin tube and tube without anticoagulant, to determine the influence of the glycolysis which occurs in vitro on the blood samples and to develop the correction factors of the delayed glycemic dosages compared to the blood glucose measurements directly after sampling.
2. Materials and Methods
This is a prospective and analytical study, conducted in Lubumbashi (DR Congo) and spread over one month (March 2017). The experimental setting was the laboratory of applications of Higher Institute of Medical Techniques of Lubumbashi. The study was conducted on 30 volunteers, 13 women and 17 men, apparently healthy, aged 19 to 44, after obtaining free and informed consent. Venous blood was collected according to good sampling practices in each of 30 subjects, fasting for 8 hours and seated, by venipuncture antecubital. Four tubes of 7 ml were thus collected by volunteer: two tubes with lithium heparin (TH), commonly used for the determination of many parameters including blood glucose and two tubes without anticoagulant (TS). For 30 consecutive working days, a different volunteer was taken daily at the same time. A tube without anticoagulant and a TH tube were immediately centrifuged for 10 minutes at 1500 g at room temperature (25˚C) to determine the reference blood glucose level. The two remaining TH and TS were respectively fractionated into three aliquots in eppendorff tubes immediately stored in a thermostated chamber at 20˚C (±1˚C), protected from light. At each of the following times (t): 1 h, 2 h, 3 h, an aliquot of each of TH and TS tubes was centrifuged for 10 min at 1500 g prior to determination of the blood glucose. A qualitative evaluation of the appearance of plasma and serum has also been performed in order to eliminate any macroscopic interference (hemolysis, turbidity). The study was carried out on the BD Vacutainer TM lithium heparin (TH) tubes and test tube without anticoagulant from cypress diagnostics. This choice was motivated by their use in daily practice in different laboratories. The glycemic determination was performed on cyanplus spectrophotometer (Cypress Diagnostics) by glucose oxidase technique. For statistical analysis, we used the hypothesis test and meanings that is to say, we realized the distribution of the difference of the means between the values of the blood glucose T0 and the blood glucose t1, t2, t3 for the tubes TH and TS at probability threshold of 5%. To determine the deferred glycemic correction factors, we used the statistical series adjustment test, i.e. we computed the linear regression line equations, the correlation and the determination coefficients between T0 and T1, T2, T3 blood glucose. The excel 2003 software was used for data processing in the form of plot lines and point clouds. All procedures herein were approved by the Research Ethics Committee of the Higher Institute of Medical Techniques of Lubumbashi, with participants giving informed consents before entering the study.
No visible interference has been demonstrated by the qualitative study of plasma and serum at different times. The blood glucose values in mg/dl are presented in Table 1, they vary at T0 for the TH tube between 64 and 123 and for the TS tube between 65 and 125 and at T3 for the TH tube between 46 and 89 and for the TS tube between 47 and 91. The statistical analysis does not show any significant
Table 1. Blood glucose values (mgl/dl) at different times for each TH and TS tube.
difference between the blood glucose values taken on the TH tube and the TS, however the difference of the means is significant at the probability threshold of 5% between the blood glucose values T0 and the blood glucose values T1, T2, T3. The equations of the regression lines, correlation and determination coefficients are presented in Tables 2-4. As the difference is not significant between TH and TS blood glucose values, these regression coefficients are applicable for both types of sampling.
From Table 2 it appears that the values of the blood glucose T0 is explained at 98% by the line Y = 1.08X + 2.5; Y is the value of the blood glucose T0 and X is the value of the blood glucose T1. The coefficient of correlation r = 0.99 shows a positive correlation between X and Y; which are directly proportional.
From Table 3 it appears that the values of the blood glucose T0 is explained at 98% by the line Y = 1.21X + 1.6; Y is the value of the blood glucose T0 and X is the value of the blood glucose T2. The correlation coefficient r = 0.99 shows a positive correlation between X and Y; which are directly proportional.
From Table 4 it appears that the values of the blood glucose t0 is explained at 98% by the line Y = 1.32X + 3.5; Y is the value of the blood glucose t0 and X is the value of the blood glucose t3. The correlation coefficient r = 0.99 shows a positive correlation between X and Y; which are directly proportional (Figure 1 and Figure 2).
Figure 1. Presentation of the results in the form of plots of the curves. Série 1: blood glucose values T0, Série 2: blood glucose values T1, Série 3: blood glucose values T2, Série 4: blood glucose values T3.The plots of the curves of the results show that all the concentrations of the blood glucose T0 are higher compared to the concentrations of delayed assays of blood glucose; T1, T2, T3.
Table 2. Linear regression line, coefficient of correlation and coefficient of determination between T0 blood glucose and T1 blood glucose.
Figure 2. Presentation of the results in the form of point clouds. Herein shows that all the points tend to approach on the same lines, i.e. the correlation between the concentrations of the blood glucose T0 and the blood glucose T1, T2, T3 is positive linear.
Table 3. Linear regression line, correlation coefficient and coefficient of determination between blood glucose T0 and blood glucose T2.
Table 4. Linear regression line, coefficient of correlation and coefficient of determination between T0 blood glucose and T3 blood glucose.
The glycolysis that occurs in vitro on the venous blood sample is known to cause a rapid decline in blood glucose. The use of an antiglycolytic agent is supposed to guarantee the stability of this parameter over time, but unfortunately antiglycolytics are problematic. A study carried out by B. Foucher et al. shows that it is only after the second hour that the anti-glycolytic effect of mono-iodoacetate is perceptible and the antiglycolytic agent; sodium fluoride creates hemolysis; this is a real problem for the screening of diabetic subjects whose measured blood glucose is close to the decision threshold when the centrifugation is not immediate  . Diagnosis criteria for diabetes mellitus are based on a fasting glucose level ≥ 11.1 mmol/l regardless of the time of day. Orally induced hyperglycemia (OIHG) remains the gold standard in the definition of diabetes (blood glucose greater than 11 mmol/l, 2 hours after OIHG) but is no longer recommended by Anaes because of its heaviness and lack of reproducibility in the same patient  . The recommended test for diabetes screening is fasting blood glucose  . Diabetes screening and diagnosis are based on a specific biological assay and threshold values. Any changes in blood glucose attributable to preanalytical delay therefore hamper interpretation especially at values close to these decision thresholds. In vitro, glucose is metabolized following the cascade of glycolysis; the addition of an anti-glycolytic agent (mono-iodoacetate, sodium fluoride or potassium oxalate) must stop this phenomenon. Our study shows a significant difference between T0 and T1, T2, T3 blood glucose concentrations and a decrease in blood glucose levels of glycemia on the TH and TS tube by about 10% per hour this means that in vitro-induced glycolysis by red blood cell affects delayed blood glucose dosing, which is why the search for correction factors in the form of regression lines linear is of great importance. The regression line y = 1.08X + 2.5 was established between the blood glucose values T0 and the glucose values T1, y being the blood glucose value T0 and X is the blood glucose value T1; the regression line y = 1.21X + 1.6 has been established between the blood glucose values T0 and the blood glucose values T2, where y is the blood glucose value t0 and X is the blood glucose value t2; the regression line y = 1.32X + 3.5 has been established between the blood glucose values t0 and the glucose values t3, where y is the blood glucose value t0 and X is the blood glucose value t3. All these lines were found with a correlation coefficient of 0.99%, which explains a correlation between the two variables. This coefficient being greater than 0, this means that these two variables are directly proportional. The values of blood glucose measured at time t = 0 is explained at 98% as coefficient of determination by the different regression lines that is to say that we are 98% sure to have made a good correction in using the linear regression line when blood glucose was not measured directly after sampling.
The established linear regression lines; y = 1.08X + 2.5 between the blood glucose values T0 and the blood glucose values T1, y is the blood glucose value T0 and X is the blood glucose value T1; the regression line y = 1.21X + 1.16 between the glucose values T0 and the blood glucose values T2, y is the blood glucose value T0 and X is the blood glucose value T2; the regression line y = 1.32X + 3.5 between T0 blood glucose values and T3 blood glucose values are likely to be used as corrective factors by health professionals when blood glucose has not been measured directly after sampling or when separation serum or plasma and figured elements of blood is not immediate. Whatever the type of tube used, the attitude to be adopted is to inform the time of sampling to allow the biologist to convert the concentrations of delayed dosing of blood glucose using established linear regression lines.
Conflicts of Interest
All authors contributed identically to the different phases of the research.
Tube TH: tube containing lithium heparin, Tube TS: tube without anticoagulant, blood glucose T0: blood glucose measured directly after sampling, blood glucose T1: blood glucose measured one hour after sampling, blood glucose T2: blood glucose measured two hours after sampling, blood glucose T3: blood glucose measured three hours after sampling.
 Vassault, A., Azzedine, M., Bailly, M., Cam, G., Dumont, G., Ekindjian, O., et al. (1986) Commission “Validation de techniques” (Commission on “Validation of Techniques”). Annales de Biologie Clinique, 44, 679-685.
 Chevillon, I., Larrose, C., Moreau, N. and Orsonneau, J.-L. (1998) Conservation des échantillons de sang avant analyse des paramètres biochimiques les plus courants. Annales de Biologie Clinique, 200-204.