And additives

Continuing in the pre-analytical phases of TDM and toxicology, it is important to think about the sample type needed and collection container. For most chemistry analyses, serum or heparinized plasma is the preferred specimen. To facilitate later steps in sample handling and testing, laboratories prefer to collect blood for these in separator or gel-barrier tubes, as the barrier formed between the serum or the plasma and the erythrocytes during centrifugation protects and stabilizes some of the measured chemistry constituents. Additionally, most laboratories have also transitioned from glass tubes to plastic for safety reasons. The previous generations of gels and other materials used in these tubes were found to adsorb many commonly monitored drugs in an unpredictable manner (5-9). Investigators found the degree of adsorption varied with the amount of drug present, the amount of blood in the tube, the environmental temperature, and the duration of exposure. There was also evidence that adsorption varied between lots of tubes in that the adsorption observed with one lot did not replicate with a subsequent lot. Because of these observations and studies, most TDM laboratories adopted the use of plain evacuated tubes containing no additives. A few laboratories adopted the use of trace metal (royal blue tops) tubes to reduce confusion among non-laboratory personnel who are involved in the collection process and likely do not appreciate the distinction between the various red- and gold-top tubes.

Over the past few years, manufacturers have introduced plastic tubes containing separator gels reportedly formulated to minimize adsorption. Unfortunately, the literature contains few reports validating the suitability of these tubes; but the studies conducted by Bush et al. (10) demonstrate the types of studies useful in validating performance of such tubes. Serum pools containing commonly monitored drugs were prepared and aliquoted into plain tubes (containing no gel) as a control and two types of gel separator tubes. The respective drug concentration was measured immediately, at 4h and at 24 h. When the results were compared with those obtained initially and with those obtained from the samples in the plain tubes, the investigators found no significant difference in the concentrations of most of the drugs for the samples in contact with the SST II gel separator tubes. Additional studies did reveal small effects using samples collected from patients receiving phenytoin or carbamazepine, two drugs for which adsorption has been shown to be a significant problem. Previous investigations had shown adsorption was more likely if the tube was partially filled or if the sample was allowed to remain in contact with the gel for extended periods of time. The investigators thus performed an extended study in which full tubes of blood were collected from patients receiving one of these two drugs. The tubes were processed and tested immediately. These results were compared with those obtained after the samples had been held at room temperature for 8 and 24 h, and stored at 4°C for 7 days. A third set of experiments were conducted simulating partial filling of tubes by placing 2 ml of serum in each of the types of tubes and the samples stored at 4°C, room temperature, and 32°C for 4, 24, and 48 h. After storage for 7 days at 4°C, the concentration of carbamazepine declined by 10% whereas that of phenytoin declined by 4%. Loss of analyte was less than 5% for the other conditions tested, well within the analytical precision of the analyses. At the time this chapter was prepared, there were few reports regarding the suitability of other reformulated barrier tubes and the previous study did not test all possible drugs or multiple lots. It is thus left to the individual laboratories to validate the claim that these new formulations do not adsorb the drugs we seek to measure. The studies outlined by Bush et al. are a reasonable approach to such studies.

Chemicals from the stoppers of the tubes, as well as chemical additives used to enhance clotting or prevent adsorption to the tubes themselves, have been found to interfere with analyses. This problem was first recognized in the 1970s when it was recognized that tris(2-butoxyethyl)phosphate (TBEP), a plasticizer, found in some stoppers, interfered with the measurement of propranolol, alprenolol, lidocaine, chlorimipramine imipramine, nortriptyline, meperidine, and quinidine (11). The mechanism of interference was interesting in that it was discovered that upon leaching into the blood sample, TBEP displaced these basic drugs from their binding sites on alpha-1 acid glycoprotein. The free drugs were postulated to then diffuse from the plasma/sera into the erythrocytes effectively reducing the total amount detected in the sera (12,13).

TBEP is no longer an issue, but problems continue to arise from interferences related to new stoppers, contaminants within the glass or plastic, or additives used enhance clotting or reduce adsorption (14-22). Methods using chromatography or mass spectrometry are particularly susceptible to some of these interferences as these materials result in extraneous peaks, co-eluting peaks, and more subtle problems of ion suppression or enhancement. For example, Murthy (14) reported a sudden appearance of an extraneous peak in a high-performance liquid chromatography (HPLC)-based method for amiodarone when a brand of evacuated tubes had been replaced by another. Unfortunately, the events that led to this problem are neither unique nor isolated. Someone outside the laboratory made the decision to switch to a different brand of tubes and did not communicate the information to the laboratory performing the testing. The potential for the collection tube to cause interferences was demonstrated by Drake et al. (15). This group tested for potential interferences from sample collection tubes for MALDI TOF mass spectrometry-based analyses by incubating 1 ml of phosphate-buffered saline (pH 7.2) in various sample collection tubes. To assure all surfaces of the tubes were considered and to maximize the ability to detect interferences, the tubes were gently rocked at room temperature for 4 h. The solutions were then processed, extracted, and analyzed. Multiple peaks were observed in the m/z range from 1000 to 3000 for solutions incubated in seven of 11 tube types.

Interferences from collection tubes extend beyond chromatography-based methods to other types of methods (19-24). For example, Sampson et al. (19) documented interference from a silica clot activator when using an ion-selective electrode-based method for measuring serum lithium. This particular report demonstrates the complexity of the interferences and the difficulty in detecting and investigating such problems. The problem was identified when a double-blind study of lithium, carbamazepine, and/or valproic acid therapy was initiated and lithium concentrations of approximately 0.1 mmol/L were reported for a patient who was not receiving lithium. The investigators found that the electrode membrane erroneously detected lithium when first exposed to samples that contained the silica clot activator, but after repeated exposure to the activator, the electrodes did not appropriately detect lithium present in patient samples. In other words, the lithium concentrations were falsely decreased. Most importantly, quality control material prepared using a bovine protein matrix was not affected and would not have detected the problem.

After considerable investigation, Bowen et al. determined that an organosilicone surfactant, Silwet L-720, was the culprit interfering with several immunoassays for thyrosine, cortisol, progesterone, thyroid-binding gobulin, and triiodothyronine (21-22). After contact with collection tubes containing this compound, results for these analytes determined using specific immunoassays were increased by as much as 11-36%. The surfactant was found to desorb antibodies from the solid phase beads used in the immunoassays leading to a reduction in the chemiluminescent signal generated and falsely increasing the apparent hormone concentration. Although the tests involved were hormonal, TDM or toxicology analyses could just have easily been involved.

Other tubes or devices, for example, microfuge tubes and filtration devices, used in the analyses should also be considered. Yen and Hsu (23) determined that contaminants from polypropylene microcentrifuge tubes yielded extraneous peaks in an HPLC electrochemical detection-based method for the measurement of antioxidants. In this case, none of the peaks interfered with the analyses as the measurements were made at multiple potentials, but as the authors cautioned, had a single potential been used for detection, interference would have been much more likely. As with several of the studies we have discussed, the extraneous peaks were not consistent across all brands tested and emphasize the lessons above that what may be perceived to be inconsequential changes to materials or methodologies may indeed be monumental.

That we continue to experience such interferences suggests that we need to continue to be diligent to detect such problems. Not only should the problems be reported to the manufacturers involved but should also be reported to the Food and Drug Administration (FDA) by accessing the MedWatch system through their Web site (www.fda.gov).

In the early days of cyclosporine measurements, small clots were found to result from the use of heparin salts and to reduce extraction efficiency. For this reason, EDTA-anticoagulated whole blood has been the sample of choice for cyclosporine and subsequent immunosuppressives found to also partition into the erythrocytes. One characteristic of cyclosporine that was not recognized for some time was its ability to adsorb to some types of plastics (25-27). I first encountered this problem when trying to automate the tedious pipetting associated with the early cyclosporine radioim-munoassays. Unfortunately, this characteristic is still not appreciated by many. Any plastic that comes into contact with these drugs should be tested before use in collection, storage, or analysis. An example of the type of study that should be conducted is seen in that reported by Faynor and Robinson (28) in which they determined that cyclosporine did not significantly adsorb to the Vacutainer PLUS evacuated tubes (Becton Dickinson Vacutainer Systems) over a 7-day period.

It may seem to be a fairly obvious statement, but lithium heparin should not be used to collect samples for lithium determinations. One practice to decrease the sample preparation time has been to adopt the use of plasma as a specimen for routine chemistry analyses. EDTA-anticoagulated blood is of course unsuitable, but lithium heparin is a popular specimen and avoids issues with sodium or potassium measurements. Which heparin salt is present within a green-top tube cannot be easily determined simply by looking at the color of the stopper. One must be able to read the contents on the label, and this may be difficult to do if the label is covered with the patient identification label. More than one laboratory has spent time investigating unexpected, elevated lithium concentrations for patients who had no evidence of toxicity only to find a lithium heparin tube had been inadvertently used for collection.

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