Every lab technician hopes to avoid errors that require retesting, increase costs, or prolong the time it takes to get results to patients. When issues do occur, it can be difficult to troubleshoot and correct the problem. As technology advances and assays become more complex, clinical laboratories need to remember that even the simplest components, like blood collection tubes (BCTs), could be responsible for discrepancies.
The laboratory testing process can be divided into three stages: the preanalytical, analytical, and postanalytical phases. As much as 70 percent of all errors made during clinical laboratory testing occur during the preanalytical phase. This important stage includes test ordering, patient preparation, and sample collection and preparation. An error during this phase can lead to challenges downstream.
BCTs play an important role in the preanalytical phase. These devices are far more complex than they look and have many added components to facilitate sample collection and preparation. Tube stoppers, stopper lubricants, clot activators, surfactants, and separator gels all contribute to the optimal separation of serum or plasma for downstream analysis. However, these components can also inadvertently introduce interfering compounds into assays.
Scientists have learned the hard way that BCTs can be a source of error in the clinical lab.
Uncovering the mystery of drug assay interference
In 1975, Cotham and Shand were studying propranolol, a beta-blocker used to treat tremors, hypertension, and many heart conditions. During these investigations, they observed lower than expected levels of this drug in patient blood samples. The scientists determined that propranolol would redistribute in favor of red blood cells (RBCs) when certain BCTs were used, leading to lower plasma concentrations of this drug.
To identify the source of this interference, Cotham and Shand collected samples in different BCTs and evaluated the concentration of added propranolol in the plasma versus RBCs. When a Vacutainer tube was used, they observed the redistribution phenomenon. However, when the stopper on this BCT was replaced with parafilm, the redistribution no longer occurred. The researchers concluded that using a green Vacutainer tube stopper led to the interference, but they did not identify the specific compound responsible.
Around the same time, New Zealand investigators Dickson et al. detected significant quantities of an unknown compound in postmortem blood samples. Using mass spectrometry, the scientists determined that the component was tributoxyethyl phosphate (TBEP), a plasticizer. They also suspected the BCTs as the source and analyzed samples from the tube stoppers with mass spectrometry. Only the stopper from the Vacutainer BCT contained TBEP. The same compound was likely responsible for the interference in Cotham and Shand’s study.
In response to the emerging literature about TBEP contamination, the manufacturer of Vacutainer introduced a new stopper that did not contain this plasticizer. This redesign ensured the Vacutainer BCTs no longer interfered with drug assays.
Discovering the source of abnormal immunoassay results
In 2005, Bowen et al. noticed that they were seeing higher than anticipated values of triiodothyronine (T3), a thyroid hormone, when performing immunoassays. Again, the researchers suspected the BCT was responsible. They compared assays performed using three different BCTs with two different analyzers. The investigators found significantly elevated levels of T3 when the Vacutainer serum separation tubes (SSTs) were used with one of the two analyzers, but not with any of the other tubes. The main difference between the tubes tested was their interior coating.
BCTs are often coated with surfactants to improve blood flow into the tubes during collection and minimize the adherence of blood cells to the tube wall. This helps reduce hemolysis and better distribute the clotting activator. The Vacutainer SST is coated with a silicon surfactant called Silwet L-720. To determine whether this coating was responsible for the increased T3 levels, researchers coated plain BCTs with different amounts of Silwet L-720 and performed immunoassays.
As expected, the presence of Silwet L-720 was significantly correlated with increased T3 concentration. A control test revealed that the immunoassay interference happened in the presence of PBS (phosphate-buffered saline) alone without any serum in the tube. The scientists hypothesized that the interference occurred because Silwet L-720 promoted the desorption of anti-T3 antibodies from the surface of the polystyrene beads used in the immunoassay. This would lower the chemilumiscent signal for T3 in the assay and falsely increase the concentration of this component.
Following these studies, the manufacturer of the Vacutainer SST decreased the amount of surfactant added to these tubes. An evaluation conducted by Wang et al. showed that the updated tube performed similarly to other BCTs and could be used interchangeably for T3 immunoassays.
Troubleshooting a new testosterone assay
While developing a LC-MS/MS testosterone assay, Shi et al. kept seeing the same interference in their results. The researchers performed their assay with different BCTs to determine whether that could be the source of the interference. Their hypothesis was correct as the interference was seen when the Vacutainer rapid serum tube (RST) was used, but not with a different type of BCT.
The researchers then tested the tube coating, stopper, and separator gel separately and pinpointed the separator gel as the source of the interference. This BCT component serves as a physical barrier between serum and clot or plasma and cells after sample centrifugation to improve separation. Unfortunately, the team was unable to adapt their assay to accommodate the Vacutainer RST. Instead, they chose to use BCTs that did not contain the interfering compound when performing future assays.
Evacuation BCTs have been evolving since their introduction in the 1940s. Despite the thorough quality control measures in place, BCTs can still be a source of preanalytical error. Every tube cannot be tested in every scenario. As a result, unanticipated interferences may be encountered during certain assays. The emergence of new tests is likely to uncover even more incompatibilities.
Clinical laboratories need to be cognizant of BCTs as a potential source of error when they encounter abnormal results. Technicians should be critical of results, especially when performing new assays. Choosing the right tube for the right test is an important practice in avoiding interference. Even still, variations between manufacturing lots could lead to unexpected findings. Implementing a regular tube-testing protocol that compares lots to each other and to glass tubes can help overcome these challenges.