Comparison of the effect of gel used in two different serum separator tubes for thyroid function tests

Abstract

Background

Selection and verification of blood collection tubes is an important preanalytical issue in clinical laboratories. Today, gel tubes are commonly used with many advantages, although they are known to cause interference in immunoassay methods. In this study, we aimed to compare SSTs of two different suppliers (Ayset clot activator & Gel and Becton Dickinson (BD) Vacutainer SST II advance) with reference tubes and evaluate the effect of storage time in terms of commonly used endocrine tests such as thyroid‐stimulating hormone (TSH), free thyroxine (fT4), and free triiodothyronine (fT3).

Methods

Fifty‐five volunteers were included in the study. Samples were taken into three different tubes and analyzed for serum TSH, fT4, and fT3 on Architect ci8200 Immunoassay System. Clinical decision levels were estimated using total allowable error (TEa).

Results

No difference was found between tubes in terms of TSH, fT3, and fT4 levels. From a statistical standpoint, TSH and fT4 levels were no longer stable during 24, 48, and 72 hours storage time periods. However, their variations were not clinically significant.

Conclusion

Ayset clot activator & Gel tubes and BD Vacutainer SST II advance tubes have comparable results with glass tube in terms of TSH, fT3, and fT4 levels on Architect ci8200 Immunoassay Systems. From a clinical standpoint, serum TSH, fT4, and fT3 concentrations may be considered as stable when storing these tubes over 72 hours.

1. INTRODUCTION

During the past decades, plastic blood collection tubes progressively replaced the glass ones.1 Today, most commonly used blood collection tubes evacuated plastic serum separator tubes (SSTs) contain separator gels that form a barrier between packed cells and serum during centrifugation. Separator gels markedly improve analyte stability and ensure storage and transport of the primary tubes.2, 3

Despite their similarity, SSTs manufactured by different suppliers vary in the materials and additives used, which may probably influence test results. Previous studies have reported that SSTs may have some interfering effects on immunoassays, but aside from the problem with therapeutic drugs, none of these effects was considered to be clinically significant.4, 5, 6 On the contrary, significant effects of gel separator tubes on total triiodothyronine test have been reported.7

To our knowledge, there has been no study in the literature considering the evaluation of Ayset clot activator & Gel tubes in immunoassay testing up to now. In this study, we aimed to compare SSTs of two different suppliers (Ayset clot activator & Gel‐Ayset Medical Devices, Adana, Turkey, and BD Vacutainer SST II advance, Becton Dickinson, New Jersey, USA) with glass tubes (reference tubes) and evaluate the effect of storage time for most commonly used endocrine tests such as thyroid‐stimulating hormone (TSH), free thyroxine (fT4), and free triiodothyronine (fT3).

2. MATERIALS AND METHODS

Fifty‐five healthy volunteers aged 20‐50 years were included in this study. All the subjects gave informed consent. The study was approved by the Local Ethics Committee of Ankara Numune Training and Research Hospital and conducted according to the revised Declaration of Helsinki (1998).

Venous blood samples were collected according to the recommendations of the Clinical Laboratory Standards Institute (CLSI) in the morning, between 8‐9 am, after a fasting period of 8 hours. Three samples from each volunteer were taken directly into one serum glass vacutainer Z tube and two serum vacuum tubes containing separator gel and clot activator with a randomized draw order. Tube types were as follows:

• Tube 1: 7‐mL BD Vacutainer Z Tube (Ref number: 367615) (Becton Dickinson, New Jersey, USA)

• Tube 2: 5‐mL BD Vacutainer SST II advance (Ref number: 367955) (Becton, Dickinson and Company Franklin Lakes, NJ, USA)

• Tube 3: 5‐mL Ayset clot activator & Gel (Ref number: 70658) (Ayset Medical Devices, Adana, Turkey)

Tubes were filled to capacity. Specimens were allowed to clot for 30 minutes at room temperature (22‐24°C) before centrifugation at 1300 g and at a temperature of 21°C for 10 minutes according to CLSI recommendations. Serum samples were analyzed within a maximum of 30 minutes after centrifugation. Samples in separator gel containing tubes were stored at 2‐8°C for 24, 48, and 72 hours (h) until reanalysis.

All samples were analyzed for serum thyroid‐stimulating hormone (TSH), free thyroxine (fT4), and free triiodothyronine (fT3) on Architect ci8200 Immunoassay System (Abbott Diagnostics, California, USA) using chemiluminescence immunoassay technique. Internal and external quality control data were checked before the study.

Statistical data were processed using PASW Statistics 18 (SPSS, Chicago, IL, USA). The conformity of variables to normal distribution was tested with Kolmogorov‐Smirnov test. The descriptive statistics of continuous variables were expressed as mean ± SD or median (min‐max). In the tube groups analysis, the presence of a statistically significant difference between the groups was examined with ANOVA for parametric and Kruskal‐Wallis test for nonparametric continuous variables. Repeated measures analysis of variance test (RMAV) and Friedman test (FT) were conducted to evaluate the differences in mean or median values of each analyte across 0, 24, 48, and 72 hours in both of the SSTs. P value <.05 was considered statistically significant. 

Statistically significant results were also analyzed for clinical significance. Total allowable error (TEa) limits were calculated from current data on within‐subject biological variation (CVw) and between‐subject biological variation (CVg) of each analyte.8

3. RESULTS

There was no statistically and clinically significant difference between three tubes in terms of TSH, fT3, and fT4 levels as shown in Table 1. According to the results of stability study, the difference for each storage time pair (0 hour‐24 hours, 0 hour‐48 hours, 0 hour‐72 hours) for Tube 2 and Tube 3 was statistically significant for TSH and fT4 results (P < .001 for each tube). fT3 results were not different till 72 hours for Tube 3 and till 48 hours for Tube 2 (P > .05), but fT3 results showed significant difference at 72 hours for Tube 2 (P = .003).

Table 1.

Comparison of the results of Tube 1, Tube 2, and Tube 3

Tests	Tube 1	Tube 2	Tube 3	P value
TSH (mIU/mL)	1.62 (0.19‐15.11)a	1.56 (0.20‐15.13)a	1.8 (0.20‐16.59)	.63c
fT4 (ng/dL)	1.05 ± 0.13b	1.04 ± 0.13b	1.03 ± 0.12	.811d
fT3 (pg/mL)	3.10 ± 0.41b	3.15 ± 0.33b	3.13 ± 0.31	.761d

Tube 1: 7‐mL BD Vacutainer Z, Tube 2: 5‐mL BD Vacutainer SST II advance, Tube 3: 5‐mL Ayset clot activator & Gel.

^aMedian (min‐max).

^bMean ± SD.

^cKruskal‐Wallis test.

^dANOVA test.

Lower and upper limits of clinical insignificant difference, calculated according to total allowable error (TEa) limits with median or mean values of TSH, fT3, and fT4, were provided in Table 2. Median or mean values of TSH, fT3, and fT4 for Tube 2 and Tube 3 were within the indicated limits.

Table 2.

Stability of thyroid hormones in both SSTs over 72 h according to clinical significance limits

n = 55	TEA %	Tube 1 (0 h)	Tube 2 (0 h)	Tube 2 (24 h)	Tube 2 (48 h)	Tube 2 (72 h)	Tube 3 (0 h)	Tube 3 (24 h)	Tube 3 (48 h)	Tube 3 (72 h)
TSH(mIU/mL)	23.7	1.62	1.56a	1.69a	1.72a	1.84a	1.80a	1.72a	1.91a	1.75a
Clin.Low		1.24	1.19				1.38
Clin.Up		2	1.92				2.22
fT4 (ng/dL)	8.0	1.05	1.04a	1.01a	1.03a	1.02a	1.03a	1.02a	1.03a	1.04a
Clin.Low		0.97	0.96				0.95
Clin.Up		1.13	1.12				1.11
fT3 (pg/mL)	11.3	3.1	3.15a	3.27b	3.29b	3.28b	3.13a	3.34b	3.30b	3.30b
Clin.Low		2.75	2.8				2.78
Clin.Up		3.45	3.5				3.48

Tube 1: 7‐mL BD Vacutainer Z, Tube 2: 5‐mL BD Vacutainer SST II advance, Tube 3: 5‐mL Ayset clot activator & Gel.

TE_A: Total allowable error, Clin.Low: lower limit of clinically insignificant difference, Clin.Up: upper limit of clinically insignificant difference.

^aStatistically insignificant difference P > .05.

^bStatistically significant difference P < .05.

4. DISCUSSION

Our results demonstrated that two different branded serum separator tubes showed no difference in terms of TSH, fT3, and fT4 levels comparing with reference tube. According to stability study, fT3 levels were stable until 48 hours for Tube 2 and 72 hours for Tube 3. Even though statistically significant, variations of serum TSH and fT4 levels remained within the total allowable error over the 72 hours storage period; thus, they were stable from a clinical standpoint.

Studies indicated that gel separator affects immunoassay test results, especially drug levels.9, 10 False negative test results were obtained because of adsorption of small molecules into gel barrier when samples were stored in separator tubes.9 In contrast, Banfi et al compared TSH, fT3, and fT4 test result of plain tubes with tubes containing thixotropic gel and did not find any difference.11 In a similar study, Kılınç et al12 did not find any difference in terms of TSH and fT4 levels except fT3 levels. Despite having small molecular structure, fT3 levels were higher in serum from gel separator tubes. In our study, there was no difference between tubes in terms of TSH, fT3, and fT4 levels. Generally, glycoprotein or peptide‐structured analytes are more likely to interact with the gel.5, 9, 13, 14 Higher TSH levels of Tube 3 may be due to cross‐reaction of substances in the assay procedure with materials of gel separator. Bowen et al indicated that total T3 levels were statistically and clinically significant and higher on Immulite 2000 analyzer (Siemens Healthineers, Germany), but no difference was found on AxSYM (Abbott Diagnostics, USA) analyzer.7 Thus, the difference of the results of gel separator and glass tubes could also be originated from the antibodies used in immunoassay techniques of different suppliers.

Stability studies of thyroid function tests in gel separator tubes have various results. Kılınç et al reported that fT3 levels were significantly different during 72 hours.12 In contrast, we did not find any difference during 72 hours for Tube 3 and 48 hours for Tube 2 in fT3 results. Schowers et al stated that fT4 levels were different during 72 hours in gel separator tubes, and the difference was clinically significant.15 Similarly, we found statistically significant difference in terms of fT4 and TSH results during 72 hours, but the difference was not clinically significant. The difference between the study results may proceed from the use of various immunoassay analyzers.

One of the limitations of our study was the reference tube, Tube 1 was not assessed for 24, 48, and 72 hours. Also, our analysis results indicate only one immunoassay technique for thyroid function tests, and the results may vary for different immunoassay analyzers using different techniques.

In conclusion, according to our results, gel containing Ayset clot activator & Gel tubes and BD Vacutainer SST II advance tubes has comparable results with reference tube in terms of TSH, fT3, and fT4 levels on Architect ci8200 Immunoassay Systems. The change in TSH and fT4 concentrations was statistically different during 24, 48, and 72 hours storage time periods, but these variations were not clinically significant. Even though without significant impact in the present conditions, comparisons of tubes by laboratories remain a preanalytical requirement to avoid the risk of false patients’ results.

Ercan M, Fırat Oğuz E, Akbulut ED, Yilmaz M, Turhan T. Comparison of the effect of gel used in two different serum separator tubes for thyroid function tests. J Clin Lab Anal. 2018;32:e22427 10.1002/jcla.22427