Preanalytical Considerations

2 PREANALYTICAL CONSIDERATIONS Toxicological analyses play a critical role in the determination of the cause and manner of death. The presence or absence of a drug and/or poison may support or refute a purported cause of death. The analytical findings may also be used to substantiate a manner of death, for example, suicide. In many instances, the measured concentration of the drug plays a pivotal role in the interpretation of the case. As critical as the analytical phase is, that is, the testing, the toxicological results can be impacted in the preanalytical phase; drug concentrations may be artificially changed due to the collection process during the autopsy, the storage conditions of the specimen and/or the storage container itself. Targeted analytes may be added to the specimen from the storage container. In many death investigations, an important question to be answered is “Was the decedent under the influence of alcohol at the time of his/her death?” Thus blood alcohol analyses are one of the most commonly requested toxicological examination performed in death investigations. In approximately one-third to one-half of these, cases are positive for ethyl alcohol. The collection of a “heart blood” sample via a blind-stick through the chest is routinely performed in some jurisdictions for the procurement of a blood sample. A blind-stick is performed by the insertion of a large gauge needle through the chest wall at an approximate 45
angle in the fourth intercostal space, targeting the ventricle of the heart. The training and skill of the specimen collector vary widely from jurisdiction to jurisdiction, some of the collections are obtained by medically trained or supervised individuals; however, there are many instances that the “heart blood” sample is collected by untrained individuals such as funeral home staff and law enforcement. A “blind-stick” collection always begs the question as to whether a valid sample is collected. Given the nature of the collection technique, the question arises as to whether or not

Postmortem Toxicology. DOI: https://doi.org/10.1016/B978-0-12-815163-1.00002-2 © 2019 Elsevier Inc. All rights reserved.

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the heart was missed and the sample originated from the esophagus, where even a small amount of gastric alcohol contaminated the sample due to agonal regurgitation. In heavily traumatized cases, the issue of the stomach and diaphragm being ruptured and thus leading to contamination by the gastric contents is a possibility. Logan and Lindholm [1] reported a case of gastric contamination of a postmortem blood sample during a blind-stick. A 45-year-old female was found dead at home. The decedent had a history of atherosclerotic coronary cardiovascular disease and noninsulin-dependent diabetes. Her physician was willing to sign the death certificate. The local corner requested that a blood sample be drawn. The specimen was drawn by local funeral home staff by a “blind-stick” through the chest cavity wall. The sample was collected in a “gray top” tube (potassium oxalate/sodium fluoride) and was submitted for toxicological analyses. The laboratory personnel described the sample as “brown-discolored and largely immobile.” The toxicological analyses revealed an ethyl alcohol concentration of 0.22 g% with an amitriptyline concentration of 6.16 mg/L and a nortriptyline concentration of 0.35 mg/L. The Coroner opined that the reported concentrations were consistent with a combined drug and alcohol overdose. The family of the decedent expressed concern over the ruling and requested that an autopsy be performed. The autopsy was performed 4 days later on the decedent. The external examination of the chest noted four postmortem needle-puncture wounds; two near the sternum and two above the left breast; suggesting several attempts at sample collection. The autopsy examination revealed that the larynx, trachea, and bronchi were over lain with a yellow-green-bile stained material and there was granular “gastric” material noted in the respiratory track, mouth, and nares. Additional toxicological specimens were collected and analyzed. Table 2.1 reveals the results of the initial toxicological tests as well as the results from the specimen (subclavian blood) collected from the autopsy. The qualitative results were the same, thus ruling out a specimen mix-up. The amitriptyline results could be explained by postmortem redistribution. The described discoloration of the autopsy “blood” suggests gastric contamination. A cytological examination of the “blind-stick” sample revealed the presence of columnar cells, ciliated columnar cells, and squamous cells and “cooked” meat fibers. This suggests the strong probability

Chapter 2 PREANALYTICAL CONSIDERATIONS

9

Table 2.1 Toxicological Results Analyte

Blind-Stick

Autopsy—SC

Ethyl alcohol Amitriptyline Nortriptyline Isopropanol Acetone

0.22 g/100 mL 6.16 mg/L 0.35 mg/L 0.01 g/100 mL 0.01 g/100 mL

0.01 g/100 mL 0.26 mg/L 0.09 mg/L 0.01 mg/100 mL 0.01 g/100 mL

that the “blood” was contaminated by unabsorbed pill fragments that were in the gastric contents. These observations suggest that the “blood” sample was contaminated by gastric contents and/or aspiration of the gastric contents and the “blind-stick” hitting the esophagus during the sampling. It is not uncommon for cases presenting to the Medical Examiner to be heavily traumatized. In such cases of extreme trauma, blood may not be available from the heart, femoral, or carotid vasculature. In these cases, blood is sometimes “scooped” from the chest cavity and submitted for analyses. This type of specimen has a high potential for contamination from the gastric contents. This usually arises from the stomach and/or diaphragm being ruptured due to the traumatic event. As a result of medical intervention, the traumatized patient may have had a chest tube inserted and blood is collected from the draining tube. The contamination issues from either specimen sources; that is, “scooped” or drainage from a chest tube are similar to those of a “blind-stick” in that the measure alcohol or drug concentration(s) may be elevated due to contamination from gastric contents. Budd [2] reported a retrospective study which he compared paired samples of chest cavity fluid and blood collected directly from the heart for alcohol content. This study covered an 8-year period and evaluated 25 cases of paired samples. There were 15 cases which had positive alcohol findings. These cases were divided into two groups; uncompromised, which some no evidence of stomach or diaphragm perforation and compromised, which some evidence of stomach/diaphragm perforation, decomposition or the sample was otherwise questionable by the examining pathologist. The results, as given in Table 2.2, suggest that a cavity blood sample is suitable for alcohol analyses if there is no compromise of the integrity of the

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Table 2.2 Comparison of Heart Blood and Cavity Blood Alcohol Concentrations Heart Blood (g%)

Cavity Blooda (g%)

Ratio

0.22 0.04 0.13 0.04 0.12 0.20 0.05b 0.11 0.26 0.08 0.10c 0.09 0.24e 0.13 0.00

0.19 0.03 0.14 0.05 0.13 0.15 0.07 0.13 0.27 0.08 0.09 0.21d 0.32 0.18f 0.03g

0.86 0.75 1.08 1.25 1.08 0.75 1.40 1.12 1.04 1.00 0.90 0.43 0.75 0.72 N/A

Mean

1.02

a

Chest/thorax/pleural blood. Spleen blood. Calculated from bile/1.4. d Stomach/diaphragm perforated. e Suspect by pathologist. f Abdominal blood. g Decomposed. Adapted from Budd 1988. b c

stomach/diaphragm or other factors that may affect the integrity of the “barriers” between the cavity and stomach, that is, decomposition. In addition to the potential for gastric contamination of blood samples collected by a “blind-stick,” the artificially raising of the apparent blood alcohol concentration, due to alcohol diffusion from the stomach to the vasculature and into the heart blood during the postmortem interval, has been suggested. To assess the validity of a postmortem heart blood sample for ethyl alcohol determinations, Plueckhahn and Ballard [3] evaluated 230 consecutive cases collecting heart and femoral blood samples for alcohol analyses. Out of the 230 paired samples, 86 of them were positive for ethyl alcohol as determined by distillation/colorimetric techniques. They found less than a

Chapter 2 PREANALYTICAL CONSIDERATIONS

0.05% probability of a statistical difference between the two measured concentrations. Their conclusion from their study was that alcohol did not diffuse at an appreciable extent into the heart blood and it was a valid specimen for analyses. Pericardial fluid will generally have an alcohol concentration up to 20% greater than the whole blood. Since this fluid may also be susceptible to alcohol diffusion from the stomach, Plueckhahn and Ballard [3] also investigated this possible artifact. They instilled alcohol into the stomach of 20 cadavers. They observed significant increases of alcohol in the pericardial fluid, whereas there were insignificant increases in heart blood alcohol concentrations. Some 45 years later practitioners, Briglia et al. [4] still debate the acceptability of heart blood specimens for the determination of alcohol. In addition to the early work of Plueckhahn and Ballard [3], data from Sunshine as well as Freireich, as referenced in Harger and Forney [5], have refuted this general notion of heart blood being unacceptable for analyses. Prouty and Anderson [6] evaluated 100 cases of paired heart and femoral blood samples to determine the distribution characteristics of ethyl alcohol from these two sampling sites. They concluded that the heart blood was a reliable specimen for testing and interpretation. However, they did observe that if there was a significant difference in the heart and femoral blood volumes collected; that the measured alcohol concentration was lower in the sample with the smaller volume as compared to the companion sample of a larger volume (femoral being lower due to the smaller sample size). This was due to loss into the headspace of the collection tube and loss once opened for sampling. Prouty and Rohrig [7] conducted a more detailed study to evaluate the relationship of sample size and/or other factors that may lead to disparate results. They evaluated a series of over 500 paired heart and femoral blood specimens. This more contemporary study utilized headspace gas chromatography for the analytical assay of ethyl alcohol. They did not note any significant differences in the measured alcohol concentrations (Heart vs Femoral) in their “normal” cases. However, in cases with medical intervention, such as placement of a central line, appreciable differences in specimen volumes and/or trauma there were noticeable differences in the measured values. Of note, in the trauma cases without medical intervention, the vast majority of the heart blood alcohol concentrations were lower than the paired femoral bloods. Agonal vomiting and subsequent aspiration is not an uncommon finding in death. The aspiration of alcohol laden

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vomitus would have the potential to raise heart blood concentration by simple passive diffusion from the lungs. Marraccini et al. [8] evaluated 307 autopsy cases, with a minimum blood alcohol concentration of 0.05 g%. Their review encompassed the premortem, agonal, and postmortem events to account for differences observed in the collect blood specimens. They found that the agonal aspiration of vomitus having at least 0.80 g% ethyl alcohol was associated with an increase in the aortic blood alcohol concentration as compared to right atrial and inferior vena cava blood. They opined that a single blood sample, in some circumstances, may not be adequate to derive an opinion of alcohol impairment. The authors suggested in addition to examining the bronchial passages for the presence of vomitus, that multiple sampling sites (e.g., vitreous) be tested to aid in the case evaluation. To investigate the impact of agonal aspiration on alcohol and drug concentrations, Pounder and Yonemitus [9] designed an experimental study using five alcohol- and drug-free human cadavers. They simulated agonal aspiration by instilling alcohol and drugs (propoxyphene and acetaminophen) into the lungs of the decedents. The bodies were held at room temperature undisturbed for 48 hours and then collected blood samples from 10 different anatomical sites. Ethanol, propoxyphene, and acetaminophen were detected in all specimens. The ethanol and drug concentrations were the highest in the pulmonary vessels (Table 2.3). The ethanol and drug concentrations in aortic blood were higher than in the left heart. The concentrations in the superior vena cava were higher than the right heart. These data suggest a direct diffusion into these vessels rather than diffusion via the pulmonary and cardiac blood.

Table 2.3 Alcohol and Drug Distribution in Simulated Agonal Aspiration Specimen

Pulmonary Vein

Pulmonary Artery

ETOH APAP PPX

58 mg% (13130 mg%) 969 mg/L (2841934 mg/L) 70 mg/L (11168 mg/L)

53 mg% (1098 mg%) 476 mg/L (141882 mg/L) 29 mg/L (7.680 mg/L)

Source: Adapted from Pounder DJ, Yonemitus K. Postmortem absorption of drugs and ethanol from aspirated vomitus—an experimental model. For Science Int 1991;51:18995.

Chapter 2 PREANALYTICAL CONSIDERATIONS

Blood and other biological specimens are routinely screened for other volatile substances in addition to ethyl alcohol. The specimen container that is used to collect and/or store the biological specimens may introduce artifacts, other volatile compounds and potentially obscure the interpretation of the subsequent analytical result. Vacutainer and other like tubes are routinely used for blood collection. These blood tubes may contain significant amounts of volatile organic compounds, such as o,m,p-xylenes and ethylbenzene. These compounds are usually introduced during the manufacturing process, most often associated with the butyl rubber stopper or the gel found in serum-separator tubes (SSTs) (Chambers et al. [10]). Streete and Flanagan [11] reported the detection of ethylbenzene and xylenes in a blood specimen collected from a patient involved in a motor vehicle accident. The sample was collected into a Sarstedt Monovette serum gel tube and stored refrigerated for more than a week prior to analysis. The tube was subsequently centrifuged and the serum assayed for ethylbenzene and xylenes. The results were ethylbenzene at 17.6 mg/L, mand p-xylene (measured as m-xylene) at 10.9 mg/L and o-xylene at 9.7 mg/L. The authors inquire of Sarstedt and confirmed that they used these solvents in the manufacturing process of the SSTs. Streete and Flanagan then obtained new tubes of a different lot number and added volatile-free blood to the tubes. The tubes were allowed to sit for 24 hours at room temperature and then centrifuged. Samples were collected at 24, 96, and 120 hours post centrifugation. Measurable concentration of ethylbenzene and the xylenes were detected in all tubes after 24 hours. The concentrations were noted to increase at 96 hours, with small but variable increases or decreases in concentrations at 120 hours. The authors noted significant differences in concentrations from lot to lot. The lowest concentrations were noted in the tubes with the earliest expiration date. They opined that this may be due to off-gassing of the solvents over time. Dyne et al. [12] reported a case of contamination by blood collection tubes by the introduction of solvents that could have resulted in an erroneous misdiagnosis of an acute poisoning. Two individuals were found obtunded in a small area where they had been utilizing toluene based glue. The hospital admission plasma toluene concentrations in the two men were 6.6 and 3.3 mg/L, respectively. Additional specimens were collected, this time in SSTs (Sarstedt Monovette) 48 hours later, while they were still hospitalized and analyzed. The toluene concentrations have appeared to increase to 11.2 and 19.2 mg/L, respectively.

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To investigate this potential contamination of the serum sample; the authors added volatile-free citrated blood bank blood to the tube and incubated at room temperature up to 101 hours. Samples were collected over time and analyzed for toluene, 1-butanol, ethylbenzene, and xylenes (m-, p-, and o-). They found that the concentrations of all targeted analytes increased over time. The mean (n 5 5) concentrations were 19, 9.9, 1.5, 0.7, and 0.4 mg/L, respectively. In addition to volatile analytes, specimen containers may introduce other compounds that may interfere with the chromatographic analyses using flame ionization or mass spectrometric detectors; for example, phthalate plasticizers (m/z 149 or 163) or common extraneous peaks that may be encountered when analyzing extracts of various bodily fluids; for example, cholesterol, methylated fatty acids. The utilization of nitrogenphosphorous detectors for gas chromatography has eliminated some of the common interferences. However, contaminates containing nitrogen or phosphorus could produce a possible interferent. Least et al. [13], Vandemark and Adams [14], and others have reported interference by the plasticizer tri (2-butoxy-ethyl)-phosphate in methylation assays for barbiturates and phenytoin. This plasticizer is commonly used in the production of rubber stoppers in some blood collection tubes. Shang-Qiang and Evenson [15] also found this plasticizer and its potential interference with chromatographic assays; they noted that it has a significant impact on the partition coefficient of 25 commonly measured drugs. Missen and Gwyn [16] reported another contaminate that is basic in chemical character and co-elutes with methadone on an OV-17 column. The compound was identified as N-isopropylN0 -phenyl-p-phenylenediamine (Fig. 2.1); via mass spectrometry (m/z 211, 40, 58, 67, and 91). This antioxidant is found in natural rubber seals used in various storage/analysis vials. Numerous practitioners have noted that in addition to adding potential interferents, the blood collection tube may also impact the stability of the drug concentration being measured. Jennison et al. [17], while monitoring amitriptyline and nortriptyline plasma levels in their psychiatric patients, noted sampling and storage conditions adversely affected the quantitation of these analytes. They noted decreases in concentration averaging 10% when the Vacutainers were refrigerated on their sides for 24 hours. To evaluate this effect, they drew samples with a glass syringe and placed them in various Vacutainer tubes. The results were variable in that the deceases in concentration ranged between 5% and 15%. Samples which were held at an

Chapter 2 PREANALYTICAL CONSIDERATIONS

Figure 2.1 N-isopropyl-N0 -phenyl-p-phenylenediamine.

elevated temperature (37
C) in contact with the Vacutainer stopper for 2.5 hours resulted in a significant loss of drug; 44% and 41% for amitriptyline and nortriptyline, respectively. The authors concluded that Vacutainer tubes, from various manufacturers, were not suitable for storage of blood specimens requiring quantitative analyses for amitriptyline and/or nortriptyline. Zetin et al. [18] studied this so-called Vacutainer effect. They collected blood samples from 17 patients who were undergoing tricyclic antidepressant (TCA) therapy (amitriptyline or nortriptyline) for the treatment of depression. Whole blood was placed into a BD Vacutainer without a stopper and covered with parafilm, with varying time from collection to centrifugation to separate the plasma. Additional whole blood samples were collected and placed into a “green-top” (heparin) BD Vacutainer tubes and others were first drawn with a syringe, then placed into a BD Vacutainer tube. A control blood was prepared by immediately separating the plasma and then extracted within 2 hours of draw. The authors concluded that the TCAs (amitriptyline and nortriptyline) were stable at room temperature for up to 3 days

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Table 2.4 Stability of Tricyclic Antidepressants Run Number of Samples

Storage Conditions

1 2 3 4 5 6

Control Room temperature Room temperature Room temperature Green vacut. Plst syringe

17 17 15 15 14 16

Time of Centrifugation (hours postdraw) 0 2 24 72 2 0.5

Total TCA Concentration Mean Valuea

SD

1.000 1.020 1.079 1.078 0.663 1.086

 0.189 0.155 0.297 0.095 0.293

a Expressed as a fraction of concentration determined by control sample. Source: Adapted from Zetin M, Rubin HR, Rydzewski R. Tricyclic antidepressant sample stability and the Vacutainer effect. Am J Psychiatry 1981;138:12478.

(Table 2.4) if the plasma did not come into contact with the stopper. Contact with the stopper, lowered the measured concentration of the tricyclic drug. This decrease in antidepressant concentration has been attributed to the plasticizer tris(2-butoxyethy)phosphate (TBEP). Vacutainer SSTs have gained widespread acceptance in the clinical arena due to the advantage of the barrier gel, which facilitates rapid separation of serum from the cellular components of blood. It also eliminates the issues of hemolysis on prolonged storage. In many instances in the investigation of a delayed death, this may be the only suitable (timewise) specimen available for toxicological analyses. Therapeutic drug monitoring has become common place in clinical medicine; however, many authors have noted a reduction in drug concentrations due to the absorption of drug into the serum-separator gel of the collection tube. Subtherapeutic concentrations of an anticonvulsant can have a significant impact on the determination of the cause of death in an individual with a seizure disorder. Parish and Alexander [19] studied the stability of phenytoin in samples collect/stored in SSTs. Samples were collected in plain red-top collection tubes and SSTs. They reported no degradation occurring in plain red-tops or SSTs that were refrigerated. The authors did note clinically significant degradation in concentration in SST tubes stored at room temperature (25
C) and at elevated temperatures (32
C) 24 hours after collection. The mean loss in concentration at room temperature was 17.9% and

Chapter 2 PREANALYTICAL CONSIDERATIONS

25.9% at the elevated temperature. They recommended that if the analyses cannot be performed within 8 hours of collection, the sample be refrigerated. Bergqvist et al. [20] studied the effect of collection tubes on patients being treated with phenytoin and two other antiepileptic drugs: phenobarbital and carbamazepine. Blood from nine patients was collected and placed either into a Venoject plain red-top tube or a SST (serum) tube. The red-top tubes were immediately poured off after centrifugation and served as the reference sample. The serum tubes were stored at 4
C with the serum in contact with the gel barrier. The tubes were aliquoted at days 1 and 7 and assayed for the target analytes utilizing standard methods. The results were compared to the reference samples (Table 2.5). They found losses in concentration of all three drugs, ranging from approximately 10% at day 1 to 20% at day 7. Dasgupta et al. [21] evaluated the stability of seven commonly monitored therapeutic agents; phenytoin, phenobarbital, lidocaine, quinidine, carbamazepine, theophylline, and salicylate in Vacutainer SST and Corvac serum separator blood collection tubes. They noted significant decreases in drug concentrations, ranging from 5.9% to 64% for all analytes but theophylline and salicylate in samples stored in the Vacutainer SST tubes. The decrease in concentration was a function of time in contact with the gel and sample volume. The most pronounced drop in concentration was observed when the sample volume was small (200500 µL) and contact with the gel barrier prolonged ( . 26 hours). The decreases were due to absorption of the drugs into the gel barrier. There were no significant changes in drug concentrations of samples stored in the Corvac serum separator blood collection tubes, except for lidocaine.

Table 2.5 Effect of Storage in Gel Tubes Antiepileptic

1 daya

Phenobarbital Phenytoin Carbamazepine

90.7 90.5 88.9

7 daysa 82.8 78.4 82.8

a Results mean data from nine patients; percent remaining. Source: Adapted from Bergqvist Y, Eckerbom S, Funding L. Effect of use of gel-barrier sampling tubes on determination of some antiepileptic drugs in serum. Clin Chem 1984;30:4656.

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The lidocaine concentrations fell from 31.5% to 72.6%, depending upon sample size, after 24 hours. The magnitude of change was related to sample size, greater change with smaller sample size in the tube. The authors were able to demonstrate the presence of the drug in the gel following chemical extract of the gel barrier with methanol. Cotham and Shand [22] postulated that the rubber stoppers in some blood collection tubes extruded a chemical that reduced the plasma binding of propranolol. The mechanism was a result of the chemical in the stopper, causing reduced plasma binding of the drug and a shift in the distribution of propranolol into the red blood cells, thus lowering the plasma concentration. They found that a reduction in plasma concentration did not occur with cardiac glycosides, phenytoin, phenobarbital, quinidine, or procainamide. The authors do suggest that this phenomenon may not be limited to propranolol, but may be seen with other highly bound basic drugs. Stargel et al. [23] noted erratic plasma concentrations of lidocaine. They investigated whether or not this was due to plasma protein binding displacement and subsequent redistribution into the red cells, as reported by Cotham and Shand with propranolol. In vitro experimentation demonstrated that the binding did decrease when the whole blood sample came into contact with the tube stopper. They opined that a chemical was released from the stopper and once in contract with the blood, it altered the plasma protein binding characteristics of the lidocaine resulting in a surreptitiously low lidocaine concentrations in serum or plasma. The authors go on to note that the plasticizer TBEP has been reported as a contaminate in plasma collected in Vacutainer tubes [24] and that TBEP has been identified as an inhibitor of drug binding to protein [25]. Therefore, they suggest that the TBEP is the compound leached from the stopper and causing the spurious results. Devine [26] confirmed that the offending chemical was TBEP. He reported that TBEP disrupts the binding of basic drugs to the carrier protein alpha1-acid glycoprotein. The author demonstrated the drugprotein binding interference caused by TBEP on lidocaine and quinidine. Lopez et al. [27] evaluated a new formulation of “red-top” (without TBEP) Vacutainer tubes. The plasma lidocaine concentrations from blood exposed to the new rubber stoppers for 1 hour were compared to those not exposed to rubber stoppers. There was no significant difference in the measured plasma concentrations of lidocaine following the 1 hour exposure. TCAs are particular prone to significant losses of analytes due to collect and storage in SSTs [28]. They tested blood samples

Chapter 2 PREANALYTICAL CONSIDERATIONS

from patient who were receiving first-generation antidepressant medications; amitriptyline, nortriptyline, imipramine, and desipramine. The study looked at the stability of these drugs in serum tubes (red-top), heparinized plasma tubes (green-top), and two types of SSTs. The concentrations of the antidepressants collected in the red-top serum tubes and plasma in the heparinized tubes were equivalent, when stored at 4
C for 4 weeks, whereas the samples collected, and spun down within 4 hours, in both SSTs demonstrated significant losses in concentration. Levy et al. [29] confirmed [28] findings of losses in TCA (amitriptyline, nortriptyline, imipramine, and desipramine) concentrations as compared to paired samples collected either in plain red-top tubes or heparinized green stopper tubes. However, their samples were spun down in less than 2 hours, which is quicker than most clinical laboratories can process samples. Evidence suggests that the longer the contact with the serum separator gel, the greater the loss of the TCA. Nyberg and Martensson [30] conducted a more extensive study; testing eight types of blood collections tubes [Venojet and Vacutainer; SSTs (gel and filter) and non-SSTs] and evaluated the loss of various TCAs (amitriptyline, imipramine, clomipramine, and their respective desmethyl metabolites); due to tube type and storage conditions, the impact of a freeze/thaw cycle. In the non-SSTs, they found significant losses of TCAs, up to 20% in the Vacutainer Royal Blue capped tubes. In SSTs, the losses were greater than 40% of the original TCA concentration. Unlike that with lidocaine, the losses were not due to redistribution into the red blood cells; but as a result of direct contact with the content and caps of the tubes. Uncentrifuged blood was found to be stable for about a day. For longer periods of time, the samples should be centrifuged for stability. Serum and plasma samples were stable at 4
C for at least 4 weeks without significant decreases in drug concentrations. The authors found no significant impact on drug concentrations from freezing, thawing and storage at 220
C. Karppi et al. [31] evaluated three different blood collection tubes and the effect of prolonged storage on the measured drug concentrations. Their study included a wide variety (41 drugs/ metabolites) of newer antidepressants, benzodiazepines, and other therapeutic agents. They found that gel SSTs did not adversely affect the measured concentrations of various antiepileptic, antibiotic, asthma, or digoxin; with absorption ranging from 0% to 5%. However, their studies found that gel tubes were not to be suitable for antidepressant and benzodiazepines, demonstrating analyte loss ranging from 5% to 30%, and up to

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40% if the samples were stored in the gel tubes after centrifugation for 24 hours. They did note for antidepressant drugs on samples stored for 3 hours showed little loss (,10%); however, after 24 hours storage the concentrations decreased anywhere from 5% to 20%. Some healthcare facilities have switched or proposed switching to plastic serum separator collection tubes instead of glass for safety reasons. Dasgupta et al. [32] evaluated the stability of a number of therapeutic drugs stored in plastic tubes and compared the results to those drugs stored in glass SSTs and glass red-top tubes. They did not observe any decrease in drug concentrations for acetaminophen, N-acetylprocainamide, amikacin, caffeine, cyclosporine, ethosuximide, methotrexate, primidone, procainamide, salicylate, theophylline, tobramycin, and valproic acid in plastic or glass SSTs. However, there was a decrease in concentrations of lidocaine, phenobarbital, phenytoin, and quinidine upon storage in either a plastic or glass SST; with increasing losses with prolonged storage and/or small blood volumes. As shown by other, the loss of drug was due to the absorption of the drug into the gel barrier. Hill et al. [33] evaluated the effect of serum separators tubes on clinical chemistries from healthy donors and hospitalized patients. They examined the most commonly tested analytes; for example, glucose, electrolytes, liver enzymes, and urea nitrogen. The comparison also involved glass SST and plastic SST tubes. The test did not show any difference between results from the gel SST tube as compared to a plain glass tube, as well as no differences were noted between the glass SST and the plastic SST tubes. The study also demonstrated the lack of any interference in the analyses from substances “leaching” from the plastic tubes. A 48-hour stability study did not show any significant difference between the target analytes, except for carbon dioxide, a significant difference was observed; which is not surprising giving the volatility of the gas. Serum methadone concentrations are frequently measured to guide dosing in patients that are nonresponsive to high-dose methadone, verify compliance, and to evaluate excessive central nervous system sedation or the expression of withdrawal symptoms. Since methadone is a weakly basic drug and frequently clinics monitoring the methadone concentrations will collect samples in SSTs, the question of whether or not collect in a SST will effect the accuracy of the analysis. Berk et al. [34] conducted a study on patients in a methadone clinic. Blood was collected in three different BD Vacutainer Tubes; plain glass, 10 mL with no additive, SST glass, 9.5 mL, and SST Plus plastic,

Chapter 2 PREANALYTICAL CONSIDERATIONS

8.5 mL. The tubes were allowed to clot, then spun down and the serum removed and frozen until analysis. They found significant reduction in methadone concentrations in both SST tubes as compared to the plain red-top tube. The mean (n 5 8) methadone concentration decrease by 24.1% comparing the glass SST tubes to the plain red-tops. The mean (n 5 7) reduction in comparing the plastic SST tubes to the plain glass tubes was 25.6% decrease in methadone concentrations. Minnick et al. [35] investigated the effect on serum element concentrations using Vacutainer tubes for collection. They analyzed for calcium, copper, iron, manganese, sodium, and zinc, over a 32-hour period. They found that zinc concentrations significantly increased over time, whereas copper and iron decreased, and there was no change in concentrations for manganese and sodium. Rodushkin and Odman [36] performed a broader assessment for the potential of trace metal contamination by devices routinely used in hospitals and clinical laboratories for the collection and storage of blood. These included various blood collection tubes, plastic pipettes and vials, gel SSTs, and stainless steel needles utilized in the collection process. The authors tested for the presence of 70 different elements. For the elements, normally found in blood/serum at concentration of 10 ng/mL or greater, the collection artifact was found to be negligible. However, for the majority of the trace and ultratrace elements the measured concentration was significantly affected. The commercially available blood collection and SSTs had the highest trace element contamination. The apparent serum concentration of several elements; for example, Al, Ba, Th, and rare earth elements were higher than expected serum concentrations in all collection tubes evaluated. Organ and tissue donation from the decedent can benefit numerous individuals and generally will not have negative impact on the medicolegal investigation of death. However, it does present some challenges to the toxicological evaluation of a case. The tissue procurement from the decedent usually results in limited blood samples being available for analyses, especially in cases of delayed death or children, who have a smaller blood volume to begin with. In the clinical setting, chain of custody is a foreign concept and usually not documented well, if at all. The anatomical site is not documented in most cases and there is an absence of some tissue all together, for example, liver and kidney. The tissue recovery process may also introduce analytes of potentially toxicological significance. Corneal procurement is

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often performed prior to an autopsy. The tissue procurement staff may wash the “field” around the eyes with isopropanol to disinfect the surrounding tissue. Vitreous fluid is often collected and submitted to the toxicological laboratory for volatile analyses. Contamination may occur from puncture of the skin or scleral surface, prior to complete drying of the isopropanol, or passive diffusion into the vitreous from the surrounding tissue. Vitreous isopropanol concentrations have been found in concentration ranges of 0.1000.200 g%, with the companion blood sample having a concentration below the detection limit of the gas chromatographic assay (0.010 g%). Another analyte that has been detected in the past from saphenous vein harvesting is papaverine. Rieders [37] reports a case where papaverine was detected in a decedent whom was thought to have died from natural causes. The papaverine concentration was 9.0 mg/L in aortic blood. Papaverine is used as a vasodilator and antispasmodic clinically, with therapeutic concentrations ranging from 0.1 to 0.4 mg/L. This finding illustrates the fact that undisclosed and undocumented chemicals or drugs administered through the harvesting process can create misleading toxicological findings and potentially a misdiagnosis of drug intoxication (Seetohul et al. [38]). In brain-dead, beating-heart donors other “artifacts” may be introduced. In brain-dead patients, a series of deleterious effects on the transplantable organ may occur. These are generally related to hemodynamic changes and physiological responses to the organ procurement [39,40]. A number of pharmacological interventions, such as vasoactive drugs, sedative/ analgesics, and paralytic agents (Table 2.6), may be utilized to manage these changes or responses to maintain the viability of the organs.

Table 2.6 Medication Used in Brain-Dead Donors Medications Ceftriazin Diltiazem Epinephrine Fentanyl

Levofloxancin Propofol Rocuronium Vasopressin

Chapter 2 PREANALYTICAL CONSIDERATIONS

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