A Comparative Evaluation of BD Vacutainer® Plus Fluoride/EDTA Tubes for Glucose & HbA1c Preservation After 24 Hours Storage at Room Temperature

VS7594-WHITE PAPER

VS7594-WHITE PAPER

A Comparative Evaluation of BD Vacutainer® Plus Fluoride/EDTA Tubes for Glucose & HbA1c Preservation After 24 Hours Storage at Room Temperature

ABSTRACT

A blood glucose evaluation comparing BD Vacutainer® Plus Fluoride/EDTA Tubes with BD Vacutainer® Serum Tubes (glass), BD Vacutainer® Lithium Iodoacetate/Lithium Heparin Tubes (glass), BD Vacutainer® Fluoride/Oxalate Tubes (glass) and BD Vacutainer® Plus Fluoride/Oxalate Tubes using specimens collected from 20 diabetic adult subjects was performed. The specimens were mixed and left for 3 and 24 hours at room temperature before being centrifuged at 1300 x g for 10 minutes. The plasma or serum was then analysed for glucose on the Roche Modular® P800 instrument using a hexokinase method. The results demonstrated that whole blood stored in the BD Vacutainer® Plus Fluoride/EDTA Tube yielded statistically and clinically equivalent glucose results to all the other tube types at initial time, and confirmed glucose stability within the BD Vacutainer® Plus Fluoride/EDTA Tube following 24 hours storage at room temperature.

As part of the same study, a HbA1c evaluation comparing BD Vacutainer® Plus Fluoride/EDTA ® ® Tubes with BD Vacutainer K3EDTA Tubes (glass) and BD Vacutainer Plus K2EDTA Tubes using specimens collected from the same 20 diabetic subjects was performed. The specimens were mixed by ten complete inversions. Measurements of HbA1c were made at initial time and after 24 hours storage at room temperature on a Tosoh A1c 2.2 instrument using an HPLC methodology. The results demonstrated that whole blood stored in the BD Vacutainer® Plus Fluoride/EDTA Tube yielded statistically and clinically equivalent HbA1c results to the BD Vacutainer® EDTA variants at initial time, and confirmed HbA1c stability within the BD Vacutainer® Plus Fluoride/EDTA Tube following 24 hours storage at room temperature.

INTRODUCTION

There are a number of enzyme inhibitor/anticoagulant combinations that are available for the stabilisation of samples for the determination of blood glucose. The choice of which inhibitor is largely dependent upon user preference. Historically, Sodium Fluoride/Potassium Oxalate has been the inhibitor/anticoagulant of choice. Other combinations have been used which may have additional benefits in some laboratory environments. All of these combinations (Lithium Iodoacetate/Lithium Heparin, Sodium Fluoride/Sodium Heparin) are available in BD glass. However, only a limited array is available in plastic (Plus). The BD Vacutainer® Plus Fluoride/EDTA Tube (BD Plus F/EDTA Tube) has been introduced to extend this array. EDTA is routinely used as the anticoagulant for the analysis of hemoglobin A1c (HbA1c) and this anticoagulant is utilised in the BD Plus F/EDTA Tube. The purpose of this study was to evaluate the suitability of the BD Plus F/EDTA Tube for measurement of both blood glucose and HbA1c.

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OBJECTIVES

To compare the performance of the BD Plus F/EDTA Tube in the determination of blood glucose at initial time with the BD Vacutainer® Serum Tube (BD Serum Tube), the BD Vacutainer® Lithium Iodoacetate/Lithium Heparin Tube (BD Iod/LiHep Tube), the BD Vacutainer® Fluoride/Oxalate Tube (BD Glass F/Ox Tube) and the BD Vacutainer® Plus Fluoride/Oxalate Tube (BD Plus F/Ox Tube) using subjects from a diabetic population.

To assess the stability of the blood glucose level in whole blood in the BD Plus F/EDTA Tube after storage at room temperature for 24 hours.

To compare the performance of the BD Plus F/EDTA Tube in the determination of HbA1c at initial ® ® time with the BD Vacutainer K3EDTA Tube (BD EDTA Tube) and the BD Vacutainer Plus

K2EDTA Tube (BD Plus EDTA Tube) using subjects from a diabetic population.

To assess the stability of the HbA1c level in the BD Plus F/EDTA Tube after storage at room temperature for 24 hours.

METHODS AND MATERIALS

The study was conducted at Saint Louis University Hospital, Saint Louis School of Medicine, USA where blood was collected from 20 subjects from a diabetic population using standard venipuncture techniques. Each subject’s phlebotomy sequence was randomized and included 2 BD Plus F/EDTA Tubes (1 for the glucose investigation and the other for the HbA1c investigation) and 1 each of the other tubes listed in Table 1.

Table 1. BD Vacutainer® Blood Collection Tubes Evaluation Tubes BD Plus Fluoride/EDTA Tube, 13 x 75 mm, 2.0 mL (two of) BD Glass Serum Tube, 13 x 100 mm, 5.0 mL Glucose Level BD Glass Lithium Iodoacetate/Lithium Heparin Tube, 13 x 75 mm, 5.0 mL Comparison Tubes BD Glass Fluoride/Potassium Oxalate Tube, 16 x 100 mm, 10.0 mL BD Plus Fluoride/Potassium Oxalate Tube, 13 x 100 mm, 6.0 mL

HbA1c Comparison BD Glass K3EDTA Tube, 13 x 75 mm, 5.0 mL Tubes BD Plus K2EDTA Tube, 13 x 75 mm, 3.0 mL

Specimen processing and handling for glucose determination

The same procedure was followed for all the different tube types in the glucose investigation, apart from the serum glass tube. The tubes were inverted 10 times immediately after collection to facilitate thorough mixing and then allowed to equilibrate upright for 3 hours at room temperature (RT) in a tube rack. The tubes were then gently inverted 2 or 3 times to ensure a uniform sample. The volume of whole blood given in Table 2 was then carefully pipetted from the tube and transferred to a BD Falcon™ Tube. This tube was centrifuged at 1300 x g (RCF) for 10 minutes

(min). The plasma was then tested for the level of glucose at the initial time (Tinitial glucose). For the BD Plus F/EDTA Tubes, the remainder of the sample in each tube was stored upright in a tube rack at RT until the 24 hours (T24) test. The tube was then gently inverted 2 or 3 times to ensure a uniform sample and treated as for the Tinitial glucose test to provide a glucose level following 24 hours storage (T24 glucose).

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Table 2. Volume of Blood Removed from Each BD Vacutainer® Tube Type in the Glucose Investigation Tube Type Volume Removed (mL) BD Plus Fluoride/EDTA Tube 1.0 BD Glass Lithium Iodoacetate/Lithium Heparin Tubes 2.5 BD Glass Fluoride/Potassium Oxalate Tube 4.0 BD Plus Fluoride/Potassium Oxalate Tube 2.0

The BD Serum Tubes were placed upright in a rack after collection and allowed to clot for 60 min at RT. The tubes were then centrifuged at 1300 x g for 10 min using a swing bucket centrifuge. The serum was carefully pipetted into respective BD Falcon™ Tubes, which were capped. Aliquots from the serum were immediately tested for Tinitial glucose.

Specimen processing and handling for HbA1c determination

For the HbA1c investigation, the BD Plus F/EDTA Tube was inverted 10 times immediately after collection to facilitate thorough mixing and then allowed to equilibrate upright for 3 hours at RT in a tube rack. The tube was then gently inverted 2 or 3 times to ensure a uniform sample. 1.0 mL of whole blood was pipetted from the tube, transferred to a microcentrifuge tube and processed for

HbA1c at initial time (Tinitial HbA1c) testing. The remainder of the sample in the capped BD Plus F/EDTA Tube was stored upright in a tube rack at RT for 24 hours. The tube was then gently inverted 2 or 3 times to ensure a uniform sample and treated as for the Tinitial HbA1c test to provide a HbA1c level at 24 hours (T24 HbA1c).

The BD EDTA Tube and the BD Plus EDTA Tubes were inverted 10 times immediately after collection to facilitate thorough mixing, then 1.0 mL of whole blood was pipetted, transferred to a microcentrifuge tube and processed for Tinitial HbA1c testing.

The glucose levels in the samples were measured by a hexokinase method using a Roche Modular® P instrument. The HbA1c levels were measured by an HPLC method using a Tosoh A1c 2.2 instrument. Table 3 lists the analytes and methodologies.

Table 3. Analytes, Methods and Analyzers Analyte Method Analyzer Glucose Hexokinase Assay Roche Modular® P HbA1c HPLC Tosoh A1c 2.2

DATA ANALYSIS

Data from all study subjects were obtained for glucose and HbA1c. The mean and standard deviation (which includes a between-subject component) were calculated for each tube type. Analysis of variance (ANOVA) procedures, followed by a pre-determined set of multiple comparisons, were used to determine whether there was a significant tube effect and to obtain the mean standard error for the confidence limits. Mean tube biases and 95% confidence limits (corresponding to 95% equivalence tests) for the various between-tube comparisons were calculated as well.

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In order to assess the equivalence between control and evaluation tubes, clinical criteria in the form of clinical acceptance limits (CALs) were established. The CAL for glucose was ±10% and for HbA1c was ±0.5%. If the value of the mean bias and the 95% confidence limit observed in the evaluation tube were within the established clinical criterion, then the evaluation tube and control tube were considered to be clinically equivalent.

As the clinical acceptance limits for glucose and HbA1c were percent-based and the data structure followed a constant CV model, the analysis was performed on the log-transformed data and the bias results back-transformed into percent biases.

RESULTS AND DISCUSSION

To assess the performance of BD Plus F/EDTA Tubes in the measurement of blood glucose and HbA1c at initial time, results were compared with a control tube. For information, comparisons with other routinely used tube types were made.

To assess the stability of the glucose and HbA1c level measurements in whole blood in BD Plus F/EDTA Tubes, the levels of these analytes in the whole blood samples stored in the tubes for 24 hours at room temperature were compared with the levels at initial time.

GLUCOSE

No bias was observed for any of the enzyme inhibitor/anticoagulant combinations used in the study as verified by comparison of the blood glucose levels in these tubes to the levels in the BD Serum Tubes at initial time.

The summary statistics for the Tinitial glucose measurements for each of the tube types are shown in Table 4.

Table 4. Summary Statistics for Tinitial Glucose Measurements for 20 Subjects from a Diabetic Population Tube Type BD Glass Analyte Measurement BD Plus F/ BD Glass BD Glass BD Plus F/ Iod/ LiHep EDTA Tube Serum Tube F/Ox Tube Ox Tube Tube Mean 158.4 160.8 154.9 157.1** 158.3 Glucose Std Deviation 66.9 69.3 69.2 76.5 69.0 (mg/dL) Minimum 75 75 71 83* 73 Maximum 315 332 323 331 325 * There was a measurement with a glucose value of “2”. As this is not a valid glucose level this observation was not used in analyses for estimating biases or means and the next lowest measurement is recorded in the table. **Mean based on 19 not 20 samples because one of the observations was not valid

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The statistics shown in Table 4 and the mean biases shown in Table 5 and Figure 1 demonstrate that whole blood collected in the BD Plus F/EDTA Tube yielded statistically and clinically equivalent glucose results to all the other tube types at initial time for this group of subjects.

Table 5. Biases and 95% Confidence Limits in Glucose Determination for Other Tube Types Compared with the BD Plus F/EDTA Tube at Initial Time Bias of the Comparative Tube Type vs the BD Plus F/EDTA Tube Analyte Bias CAL BD Glass Serum BD Glass BD Glass F/Ox BD Plus F/Ox Tube Iod/LiHep Tube Tube Tube Mean 1.2% -3.1% 1.1% -0.5% Glucose (±95% confidence ±10% limits) (-2.5%, 5.1%) (-6.7%, 0.6%) (-2.7%, 5.1%) (-4.2%, 3.3%)

Figure 1. Mean Bias Plots for the Tinitial Glucose Determination (Clinical Acceptance Limit of ±10%)

BDPAS 05-016 Part A: Diabetic Population (Initial Time): Glucose Bias withBias with 95% Confidence confidence Limits limits 10 10%

5 Bias (%) 0

-5

-10 -10%

G G P G l GlassP Seruml - P la PlusP NaF/KOxlu - – a a s lu s lu s l s P NaF/EDTAs plus s NaF/EDTAu F plus l s s s s u I S Glass NaF/KOx F - /O Glass Li Iod/Lis o Hep - F e F / F d /E r /E O /E x F / u NaF/EDTA plusx NaF/EDTA/ plusL D m D D – E i T T – T D H A – A A T e A p

The summary statistics for the Tinitial and T24 glucose measurements for the BD Plus F/EDTA Tube are shown in Table 6.

Table 6. Summary Statistics for the Tinitial and T24 Glucose Measurements for 20 Subjects from a Diabetic Population for the BD Plus F/EDTA Tube Time Analyte Measurement Tinitial T24 Mean 158.4 157.6 Glucose Std Deviation 66.9 67.7 (mg/dL) Minimum 75 73 Maximum 315 320

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The statistics shown in Table 6 and the mean bias shown in Table 7 demonstrate that for this group of subjects the BD Plus F/EDTA Tube yielded statistically and clinically equivalent glucose results following storage of whole blood in the tube at RT for 24 hours when compared with the level at initial time.

Table 7. Bias and 95% Confidence Limits in Glucose Determination for the BD Plus F/EDTA Tube

at T24 Compared with Tinitial

Analyte Bias CAL Bias of T24 vs Tinitial Mean -0.7% Glucose ±10% (±95% confidence limits) (-5%, 3.7%)

HEMOGLOBIN A1c (HbA1c)

The summary statistics for the Tinitial HbA1c measurements for each of the tube types are shown in Table 8.

Table 8. Summary Statistics for Tinitial HbA1c Measurements for 20 Subjects from a Diabetic Population Tube Type Analyte Measurement BD Plus F/EDTA Tube BD EDTA Tube BD Plus EDTA Tube Mean 7.26 7.26 7.25 HbA1c Std Deviation 1.77 1.75 1.72 (%) Minimum 4.9 4.9 4.9 Maximum 11.4 11.4 11.4

The statistics shown in Table 8 and the mean biases shown in Table 9 and Figure 2 demonstrate that whole blood collected in the BD Plus F/EDTA Tube yielded statistically and clinically equivalent HbA1c results to all the other tube types at initial time for this group of subjects.

Table 9. Biases and 95% Confidence Limits in Tinitial HbA1c Determination for Other Tube Types Compared with the BD Plus F/EDTA Tube Bias of the Comparitive Tube Type vs the BD Plus F/EDTA Tube Analyte Bias CAL BD Glass EDTA Tube BD Plus EDTA Tube Mean 0.01% -0.01% HbA1c (±95% confidence ±0.5% limits) (-0.18%, 0.19%) (-0.19%, 0.17%)

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Figure 2. Mean Bias Plots for the Tinitial HbA1c Determination (Clinical Acceptance Limit of ±0.5%)

BDPAS 05-016 Part A: Diabetic Population (Initial Time): HBA1C Bias withBias with 95% Confidence confidence Limits limits 0.5 0.5%

Bias (%)

0.0

-0.5 -0.5%

K3EDTA GlassGlass - EDTANaF/EDTA – plus Glass EDTA – Plus F/EDTA Plus F/EDTA K2EDTA Plus - NaF/EDTA plus

The summary statistics for the Tinitial and T24 HbA1c measurements for the BD Plus F/EDTA Tube are shown in Table 10.

Table 10. Summary Statistics for the Tinitial and T24 HbA1c Measurements for 20 Subjects from a Diabetic Population for the BD Plus F/EDTA Tube Time Analyte Measurement Tinitial T24 Mean 7.26 7.25 HbA1c Std Deviation 1.77 1.72 (%) Minimum 4.9 5.0 Maximum 11.4 11.4

The statistics shown in Table 10 and the mean bias shown in Table 11 demonstrate that for this group of subjects the BD Plus F/EDTA Tube yielded statistically and clinically equivalent HbA1c results following storage of whole blood in the tube at RT for 24 hours when compared with the level at initial time.

Table 11. Bias and 95% Confidence Limits in HbA1c Determination for the

BD Plus Fluoride/EDTA Tube at T24 Compared with Tinitial

Analyte Bias CAL Bias of T24 vs Tinitial Mean -0.01% HbA1c ±0.5% (±95% confidence limits) (-0.16%, 0.14%)

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CONCLUSIONS

The BD Vacutainer® Plus Fluoride/EDTA Tube had statistically and clinically equivalent results when compared with the BD Vacutainer® Glass Serum Tube, the BD Vacutainer® Glass Lithium Iodoacetate/Lithium Heparin Tube, the BD Vacutainer® Glass Fluoride/Oxalate Tube and the BD Vacutainer® Plus Fluoride/Oxalate Tube for the determination at initial time of blood glucose in this diabetic population using the Roche Modular® P instrument with a hexokinase method.

The BD Vacutainer® Plus Fluoride/EDTA Tube had statistically and clinically equivalent results for the measurement of blood glucose level following storage of the whole blood sample in the tube at room temperature for 24 hours when compared with the blood glucose level at initial time.

The BD Vacutainer® Plus Fluoride/EDTA Tube had statistically and clinically equivalent results ® ® when compared with the BD Glass Vacutainer K3EDTA Tube and the BD Vacutainer Plus

K2EDTA Tube for the determination at initial time of hemoglobin A1c (HbA1c) in the blood of this diabetic population using the Tosoh A1c 2.2 instrument with an HPLC method.

The BD Vacutainer® Plus Fluoride/EDTA Tube had statistically and clinically equivalent results for the measurement of HbA1c following storage of the whole blood sample in the tube at room temperature for 24 hours when compared with the HbA1c level at initial time.

Whenever changing any manufacturer’s blood collection tube type, size, or storage condition for a particular laboratory assay, the laboratory personnel should review the tube manufacturer’s data and their own data to establish/verify the reference range for a specific instrument/reagent system. Based on such information, the laboratory can then decide if a change is appropriate.

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