Insignificant interference of Elevit in pregnant women serum samples with HBsAg immunoassay on Sysmex

Abstract

Background

The pregnant women often take Elevit as the multivitamin supplement which has a substantial amount of biotin that might potentially interfere with the HBsAg immunoassay performed by the prevalent Sysmex system in clinical laboratories. We therefore wanted to determine this, so that the therapeutic intervention on the hepatitis B virus infection during pregnancy and birth would not be missed.

Methods

Elevit was both serially diluted in vitro and orally taken by healthy volunteers whose blood samples were then taken at different time points. All samples were added to a serum sample with a known result of HBsAg and then measured by Sysmex. The Abbott immunoassay system was used as the control as it involves no streptavidin‐biotin binding in the reagent set. Besides, the HBsAg results were compared between the pregnant women taking or not taking Elevit.

Results

Biotin at 25 ng/mL in the Elevit started to suppress the HBsAg and reached about 50% suppression at 100 ng/mL on Sysmex. In the volunteers, biotin reached the peak concentration at 2 hours. However, their blood samples showed no suppression on the HBsAg detection by Sysmex. In samples from pregnant women who took Elevit, the HBsAg results by Sysmex were highly correlated with those by Abbott (R ² = 0.96). Comparison of the results from Sysmex between the age‐ and pregnancy‐matched females with and without Elevit intake showed no difference.

Conclusion

Elevit intake in pregnant women shows no significant interference with HBsAg immunoassay on Sysmex.

1. INTRODUCTION

The HBsAg screening in the pregnant women is a routine, and is essential for the possible therapeutic intervention of Hepatitis B virus infection in the newborns.1, 2, 3, 4 This assay is often performed on the Sysmex system, one of the prevalent automated immunoassay analyzers in clinical laboratories.5, 6, 7 In this system, the biotinylated‐antibody is first added to the serum sample to bind HBsAg and then captured by the streptavidin‐conjugated magnetic beads for further detection.

It is known that high concentration of endogenous biotin in the specimen might compete with the biotin conjugated on the antibody in immunoassays (Figure 1A), causing false results.8, 9 Elevit, a multivitamin supplement that contains a substantial amount of biotin,10, 11 is often orally taken by pregnant women for possible health improvement.

Figure 1.

Elevit interferes with HBsAg detection by Sysmex. A, The working principle of HBsAg assay on Sysmex. The biotinylated antibody (anti‐HBsAg) is added to the serum sample to bind the HBsAg. The endogenous biotin competes with the biotin on the antibody for the streptavidin‐conjugated magnetic beads. B, Serially diluted Elevit in the sample inhibits HBsAg detection on Sysmex. In the Abbott immunoassay system, the anti‐HBsAg is directly conjugated to the beads with no biotin‐streptavidin involved. The discrepancy of the HBsAg quantitation results by Sysmex and by the Abbott assay kits is due to their independent HBsAg calibrators. One way ANOVA followed by Tukey post‐hoc test were used (n = 3)

Therefore, it is possible that the HBsAg immunoassay of the blood samples from pregnant women might be affected by the Elevit that they take, giving rise to false negative results that lead to a serious misdiagnosis in clinic. We therefore investigated this in the current study.

2. MATERIAL AND METHODS

2.1. Serial dilution of Elevit and the interference with HBsAg detection

One tablet of Elevit (Bayer Health Care, Berlin, Germany, contained 0.2 mg biotin) was dissolved in 100 mL saline to make 2 μg/mL biotin, from which 100 μL was added to 0.9 mL serum with positive HBsAg to make 200 ng/mL. Meanwhile, 1 mL saline was added to 9 mL of the same serum as the diluent. A 0.5 mL of the 200 ng/mL HBsAg serum sample was then taken to make serial 2‐fold dilutions in the diluent serum. This serial dilution protocol did not use saline directly as the diluent because the biotin might lose at low concentrations due to lack of carrier substances in the solution.

2.2. Immunoassay of HBsAg by Sysmex and Abbott systems

The two systems use their own HBsAg calibrators for their reference curves independently, so their result values were therefore not directly equal. Both follow the routine sandwich immunoassay procedures. The major difference is that Sysmex uses biotinylated antibody and the streptavidin‐coated magnetic particles to capture the HBsAg, while Abbott uses the antibody directly conjugated to beads.

2.3. Oral intake of Elevit, blood collection, and biotin measurement in healthy volunteers

Three volunteers (two females and one male, 35, 40, and 41 years old) were recruited. Initially blood samples were collected in the morning after the food fasting overnight (0 hour), and then each volunteer took one tablet orally. Then, more blood samples were taken at 2, 4, and 6 hours. The biotin level in the serum was measured with the commercial kit (EBD0325; Elabscience, Houston, China) which is a competitive ELISA. All serum samples were mixed with an HBsAg‐positive sample and then analyzed by both Sysmex (Kobe, Japan) and Abbott (Chicago, IL, USA).

2.4. Data processing and statistical analysis

The quantitative results (unit/mL) were directly taken, or were normalized for analysis. One way ANOVA followed by Tukey post‐hoc test and Student's t test were used.

3. RESULTS

The in vitro‐serially diluted Elevit started to suppress the HBsAg measurement at 25 ng/mL of biotin, and reached about 50% suppression at 100 ng/mL of biotin in the Sysmex immunoassay system. In contrast, no suppressive effect was observed in the Abbott which uses antibody directly cross‐linked to magnetic beads (Figure 1B).

In the three volunteers who were HBsAg‐negative, the biotin reached peak concentration (10.0, 21.4, and 8.4 ng/mL, respectively) at 2 hours after they orally took Elevit, and then reduced slowly at 4 and 6 hours. However, none of their blood samples at any collection time point showed suppressive effect on the HBsAg immunoassay on Sysmex (Figure 2A). Statistical analysis of the normalized results of these three individuals showed no significant difference (Figure 2B).

Figure 2.

In‐vivo effect of Elevit on HBsAg detection by Sysmex. A, Effect of Elevit in healthy volunteers on the HBsAg immunoassay. Three volunteers orally took Elevit and their blood samples were collected at the indicated time points. Biotin was measured, and then the samples were mixed with an HBsAg‐positive serum sample. B, No interference was observed in the blood samples after oral intake of Elevit. C, Correlation of HBsAg assay results by Sysmex and by Abbott in serum samples from pregnant women. D, Further comparison of these samples with age‐ and pregnancy‐matched females with no intake of Elevit. Each result of HBsAg from Sysmex was divided by that from Abbott. If the Sysmex results were interfered by the biotin from Elevit, then the ratio (Sysmex/Abbott) should be smaller, which was not seen here

We also collected about 50 serum samples from pregnant women who took one tablet of Elevit daily. The HBsAg immunoassay was repeated immediately on Abbott after they showed positive HBsAg results on Sysmex. Although the results from Sysmex were largely lower than those from Abbott, they showed very high correlation (R ² = 0.96), indicating the discrepancy of the results by these two systems is very likely from the use of independent HBsAg calibrators in their reference curves, but not from the biotin interference issue (Figure 2C).

We further compared the results between the pregnant women with Elevit intake and those age‐ and pregnancy‐matched females who had no intake of Elevit or other vitamin‐containing supplements, by taking the ratio of results of Sysmex and Abbott (Sysmex/Abbott) for analysis. If biotin had interfered with the HBsAg detection on Sysmex, then the Sysmex/Abbott ratio should be smaller. However, this was not seen in these two groups (Figure 2D).

4. DISCUSSION

In this study, we investigated whether Elevit possibly interfered with the detection of HBsAg on the Sysmex immunoassay system. Although Elevit showed a suppressive effect in vitro, this was not seen in vivo in healthy volunteers or the pregnant women.

The most possible reason is that the biotin from Elevit cannot reach the concentration high enough to compete with the biotin on the antibody for the streptavidin‐beads. The in vitro‐titrated Elevit started to suppress the HBsAg detection slightly on Sysmex at 25 ng/mL, but the peak biotin concentration in blood after Elevit intake was only about 20 ng/mL.

Because each Elevit tablet only contains 200 µg biotin and the total plasma volume in an adult is 3 L approximately, the theoretical peak concentration of biotin after the intake of Elevit is no more than 66.7 ng/mL, which explained the actual peak concentration (10‐20 ng/mL) in this study. Besides, the normal range of the endogenous biotin in adults is below 1 ng/mL.12 Therefore, the oral intake of Elevit is generally insufficient to exert interference on the detection of HBsAg by the Sysmex system.

Besides, because the blood sample collection from the pregnant women is often performed in the morning after an overnight of food fasting, which has far passed the peak time point, therefore the interference from Elevit taken in the last day is minimal. We therefore conclude the intake of Elevit in pregnant women is unlikely a problem for the screening of their HBsAg in the blood samples.

Ning M, Chen Y, Zheng Q, Jia J, Bai B. Insignificant interference of Elevit in pregnant women serum samples with HBsAg immunoassay on Sysmex. J Clin Lab Anal. 2019;33:e22725 10.1002/jcla.22725