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Laboratories and distributors received a notification on 30 May 2019, of a voluntary nationwide recall of a specific lot of blood collection tubes from one of the world’s largest manufacturers. According to the recall notice, a small portion of the tubes already shipped to end-users was lacking the required chemical preservatives. Specifically, the product was 16 × 100 mm glass evacuated grey top tubes containing preservative (sodium fluoride, 100 mg) and anticoagulant (potassium oxalate, 20 mg) designed for a 10 mL blood draw. There were 247,000 tubes in the particular lot with an initial distribution date of 31 August 2018. On 12 June 2019, the manufacturer issued an amended notice, announcing that a total of 300 tubes did not contain any preservative and anticoagulant. Nine days later the manufacturer issued another letter stating that 199 of these 300 tubes were received by a single client and had already been returned to the manufacturer. Furthermore, the laboratory of one of the authors identified 73 tubes without additives simply by making a visual inspection and these were returned to the manufacturer. This leaves just 28 remaining unaccounted for tubes, equating to just ~ 0.01% of the total batch at the time of writing. There also remains the possibility that a single facility received the remaining 28 tubes, discarded them, and did not communicate this to manufacturer. The root cause was identified and described in the recall notice as a manufacturing issue and was immediately corrected. This commentary aims to describe the issues pertaining to any medicolegal casework potentially associated with the recalled lot. Specifically, the likelihood of these tubes being used in forensic casework, and if so, the impact, if any, this would have had on the interpretation of results if tubes without preservative and anticoagulant had been used for determination of ethanol and other toxicological substances. Issues With the Recall MemorandumsPer the manufacturer recall memoranda dated 12 and 21 June 2019, “Based on publicly available scientific literature, in cases where a sample is processed without the additive in the tube, testing has yielded reliable results if the samples were stored at room temperature for no longer than two days. If the sample was stored for >2 days, the result for blood alcohol determination might not be accurate (either falsely low or falsely high).” The recall memorandum cited “Tietz Clinical Guide to Laboratory Tests” by Wu as the source of the statement (1). This guide explained that unopened blood specimen tubes without sodium fluoride (NaF) are stable at 25, 5 and −15°C for ~2 days, 2 weeks and 4 weeks, respectively. However, no part of this reference source specifically supported the recall statement “alcohol determination might not be accurate either falsely low or falsely high.” To help clarify the memorandum statement and how it relates to either living or decedent casework, the authors of this communication contacted the manufacturer and a representative provided further information including a paper by Kaye (2). This article does contain information about ethanol stability and states “We have found that blood without sodium fluoride is reliable at: room temperature (25 C) for about two days; refrigeration (5 C) for about two weeks; freezer (-15 C) for about 4 weeks.” Kaye continues, “These approximations are based partly on work by Cardona on 172 autopsies and partly on unpublished work.” Furthermore, Kaye stated, “Without fluoride, the BAC may rise to a maximum in about 15 days and then fall. With fluoride, when blood is kept beyond the limits of storage, although lower alcohol levels are often found, higher levels may also be seen.” To support this statement, Kaye referenced a paper by Bogusz et al. (3) from 1970 entitled “Studies on the formation of endogenous ethanol in blood putrefying in vitro.” These investigators stored unpreserved postmortem blood at room temperature and measured the formation of ethanol under anaerobic conditions for various periods of time. The blood ethanol concentration initially increased up to the fifth day before stabilizing and reaching the highest concentrations on the 15th day, followed by a slow decline. Two important conclusions can be drawn from the customer notifications about recall of the evacuated tubes. First, the manufacturer did not provide appropriate updates nor complete or relevant information in their recall memorandums regarding blood ethanol concentrations. Secondly, all cited studies exclusively involved postmortem specimens. Therefore, the recall statements were not relevant to blood samples collected from living subjects under sterile conditions (e.g., during drink- or drug-driving cases). Safe-Guarded by Good Forensic PracticeIn a forensic setting, the tubes involved in the recall are typically used in certain blood collections at autopsy for medicolegal death investigations and by phlebotomists or other medical professionals for law enforcement investigations (4, 5). There are several ways evacuated tubes are distributed: typically, laboratories will receive batches of evacuated tubes from vendors and then distribute them to phlebotomy service providers and other end-users as required; or those parties receive the tubes directly from the manufacturer or its various distributors; or collection kits are prepared by a third-party vendor and distributed to laboratories or phlebotomists. With a single client having received two-thirds and another single laboratory receiving a quarter of the inappropriate tubes, it appears unlikely that the 28 unaccounted for tubes were distributed sporadically. While the possibility of a laboratory unknowingly having used one or more of these tubes was unlikely, many laboratories promptly alerted criminal justice stakeholders in their respective jurisdictions about the recall notification. Additionally, as good forensic practice, some laboratories performed internal investigations to assess the possibility of receiving any affected tubes. In an abundance of caution, many laboratories and phlebotomy service providers discarded stocks of tubes within the lot even if they appeared normal and replaced them with tubes having a different lot number as offered by the manufacturer. Visualizing clotsThe absence of additives would be visually indicated by the lack of whitish colored powder in the evacuated tube prior to blood collection. Additionally, without potassium oxalate (anticoagulant), blood samples would start to clot following collection (6). Certain medical conditions that decrease clotting may differently affect normal clotting mechanisms. However, such medical issues are typically questioned and disclosed to a phlebotomist for safety reasons, sometimes requiring individuals to certify before a blood draw that they have not been diagnosed with hemophilia or are not being medicated with anticoagulant drugs. Additionally, as per legal necessity, good clinical practice and/or standard operating procedures, phlebotomist service providers typically inspect tubes prior to use and verify the inclusion of a white powder (7). Laboratories may also perform checks for additives prior to the tubes being distributed to phlebotomists in the blood specimen collection kits or otherwise. Furthermore, upon receipt of blood samples for analysis, forensic laboratories routinely inspect and document any unexpected observations or unusual conditions prior to and/or during analyses (e.g., clotted sample, volume and color of the blood). Finally, if tubes absent of additives managed to be received, distributed, used and submitted as evidence without notice, clotted blood samples would be challenging or impossible to pipette and dilute with internal standard without notice, particularly for ethanol determinations where thinner pipette tips are typically utilized. Internal investigationsThe notice of a tube recall issued by the manufacturer also indicated that after collection, it would not be possible to determine whether or not the additive was present. However, because of clotting and the routine visual inspection of the blood specimens on arrival at the laboratory before analysis, the lack of anticoagulant and also preservative would be obvious. This makes it very unlikely that such a specimen would be collected, pass inspection and analyzed without noticing a problem and reacting accordingly. A laboratory may even choose to further investigate all known instances when defect tubes might have been initially used without noticing the formation of a clot or other irregularities. In addition to the usual documentation of observations and conditions of the specimen described above, another round of inspections of these samples can be performed. This kind of inspection can be done even after the blood specimens have been stored for longer periods of time since collection, to ascertain whether they are in normal condition or clotted (6). Anticipated EffectsNonetheless, if a blood specimen collected in a tube without anticoagulant and preservative had been analyzed, with or without noticing a clot; inferences can still be drawn on how this affects the reliability of ethanol and drug concentrations in routine casework. Ethanol (alcohol)As mentioned for postmortem blood samples, the manufacturer states that if the specimen had been stored for >2 days, the blood–alcohol concentrations (BAC) might be either falsely low or falsely high (1). In regards to blood specimens from living subjects, most scientific literature has demonstrated that BACs are not significantly affected by the lack of NaF preservatives when collected in an aseptic manner and stored refrigerated or for 2 weeks at room temperature (8–10). Another study demonstrated that for ~48 h after sampling, there was no ethanol detected in unpreserved antemortem blood when stored at 4 or 20°C (11). At elevated storage temperatures ethanol concentrations were found to decrease even when NaF preservative was present (9). Decreases in BAC were also noted when blood samples with a fluoride preservative (1%) were kept in a refrigerator for several months or years before reanalysis (12–14). Overall decreases in BAC were also observed in unpreserved blood (15). Another study showed that with or without NaF preservative, the concentration of ethanol after 10 days of storage at room temperature or refrigerated yielded results 5 to 9% lower than bloods analyzed within 24 h (10). Furthermore, the standard practice in drink-driving cases in Germany is to take blood from suspects into empty (unpreserved) evacuated tubes, and on arrival at the laboratory, the serum portion is obtained by centrifugation. The quantitative analysis of ethanol is performed on this unpreserved serum for legal purposes (16). Although not in grey-top collected whole blood, this demonstrates the accepted use of ethanol analysis from an unpreserved blood source and use in a legal context. Studies have shown that in blood inoculated with a common yeast (Candida albicans) and glucose, ethanol was produced if there was no NaF present (15, 17, 18). However, one of these studies found extremely low production of ethanol when blood from living people was used (17). Another failed to observe production of ethanol in blood if the unpreserved specimen was not fortified with additional glucose (18). Moreover, one study showed that in yeast-inoculated preserved and non-preserved blood, there were no increases in BAC for ~29 weeks storage at different temperatures (15). For both the safety of the individual, and for mitigation of any microbe contamination, blood collection is typically performed by phlebotomists utilizing sterile techniques and equipment. In forensic casework, use of non-alcohol swabs to disinfectant the skin and aseptic equipment is legally mandated (7). For these reasons, the perceived need for a fluoride preservative in the evacuated tubes when blood samples are taken from living people under sterile conditions might be exaggerated. Sampling blood from decedents is a different scenario because of increased opportunity for microbe contamination and/or bacterial growth during autolysis (19). However, 1% NaF preservative was effective in inhibiting production of ethanol in postmortem specimens, even when blood glucose concentrations were made artificially high (18). Ethanol production in unpreserved postmortem blood was highly variable depending on temperature and duration of storage prior to analysis (20). More ethanol was generated when specimens were stored at elevated temperatures rather than typical room temperature (20–30°C), although no ethanol production was observed when bloods were frozen (20). Methods are available to identify the source of ethanol in postmortem blood via the analysis of alternative specimens, such as vitreous humor and/or urine (21). In addition, a review of the deceased’s medical history for uncontrolled diabetes and/or hyperglycemia may also aid in differentiating postmortem synthesis of ethanol from antemortem ingestion (21). To reiterate, if an anticoagulant is not present in the collection tube then freshly drawn blood will begin to clot (6). Most jurisdictions in the USA require specimens of whole blood for determination of ethanol in impaired driving cases (22). Therefore, if blood is clotted, the impact of specimen homogenization on the interpretation of analytical results is important to consider. When clotted specimens were analyzed following the intentional homogenization of the clot using a tissue grinder, the maximum BAC deviation that resulted was within typical uncertainty and acceptable precision ranges (6). However, this process requires transfer of the entire sample to an additional container, heat is generated in the treatment process and this may lead to some losses of ethanol (6). Other drugsThe impact of a lack of preservative in the stability, analysis and interpretation of drugs other than ethanol also deserves consideration. From reviewed literature, drug concentrations in both antemortem and postmortem blood tend to decrease over time without preservatives. Most notably, cocaine concentrations have been shown to undergo considerable degradation over time in unpreserved blood (23–29). Sodium fluoride mitigates such degradation by blocking the process of cholinesterase metabolism and hydrolysis of cocaine to its metabolites, benzoylecgonine and ecgonine methyl ester. However, even these products showed evidence of further hydrolysis to ecgonine (23). In casework involving heroin, the unique metabolite and marker of its use, 6-acetylmorphine, is rapidly degraded in unpreserved blood (30). The inactive metabolite of delta-9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC glucuronide, was also shown to hydrolyze to another inactive metabolite, 11-nor-9-carboxy-THC in samples collected without preservatives (23). These studies lead to the conclusion that concentrations of some active drugs can decrease in blood lacking NaF preservatives, although the concentrations of their inactive metabolites might increase. ConclusionIn summary, the possibility of reporting falsely high blood ethanol concentrations in gray-top tubes without anticoagulant and preservative is overwhelmingly low when samples are taken from living persons. Typical phlebotomy practices coupled with extensive observations, documentation and standard operating protocols commonly used in forensic laboratories are among the safeguards to ensure specimen integrity. Standard refrigeration of specimens (4°C) have been shown to mitigate any increases in ethanol concentration, even in postmortem blood. Concentrations of ethanol, and many other drugs, actually decrease during storage without a fluoride preservative. While anticoagulant is required to maintain the integrity of a whole blood specimen, the optimal concentration of fluoride preservative or even its scientific necessity remains an open question when specimens are drawn from living persons. However, for convenience and the advantage of working with just one type of tube for both postmortem and antemortem specimens, the authors of this communication are not recommending laboratories change their current practices. Only the manufacturer can respond to issues or questions about the manufacturing process, such as the percentages of tubes originally affected, the small number of recalled tubes remaining unaccounted for and the root causes of the recall. However, in typical criminal justice cases when blood is taken from living subjects, this lack of anticoagulant and preservative in the tubes would be largely noticeable and inconsequential, respectively. We conclude that the recall notification and the underlying warning about risk of false blood ethanol concentrations were misleading. References4. , , , , , , , , , , , , et al. () Collection of biological samples in forensic toxicology .Toxicology Mechanisms and Methods , , –.5. , , , , , () Guidelines for collection of biological samples for clinical and forensic toxicological analysis .Forensic Sciences Research , , –.6. , , , () The accuracy of blood alcohol analysis using headspace gas chromatography when performed on clotted samples .Journal of Forensic Sciences , , –.10. , , , , , , , () Comparison among plasma, serum, and whole blood ethanol concentrations: impact of storage conditions and collection tubes .Journal of Analytical Toxicology , , –.12. , () Are changes in blood-ethanol concentration during storage analytically significant? Importance of method imprecision .Clinical Chemistry and Laboratory Medicine , , –.14. , , , () Decreases in blood ethanol concentrations during storage at 4°C for 12 months were the same for specimens kept in glass or plastic tubes .Practical Laboratory Medicine , , –.15. , , , , , () Pre-analytical factors related to the stability of ethanol concentration during storage of ante-mortem blood alcohol specimens .Journal of Forensic and Legal Medicine , , –.17. 18. , , , , , , , , , , , () Ethanol production by Candida albicans in postmortem human blood samples: effects of blood glucose level and dilution .Forensic Science International , , –.19. , () Possible sources of ethanol ante- and post-mortem: its relationship to the biochemistry and microbiology of decomposition Journal of Applied Bacteriology , , –.20. , , , , , () Postmortem production of ethanol in different tissues under controlled experimental conditions .Journal of Forensic Sciences , , –.24. , , , , , , , () In vitro stability of cocaine in whole blood and plasma including ecgonine as a target analyte .Therapeutic Drug Monitoring , , –.26. , , , , , () The role of ecgonine methyl ester in the interpretation of cocaine concentrations in postmortem blood .Journal of Analytical Toxicology , , –.28. , , , , , , , , , , , , et al. () The effect of sodium fluoride preservative and storage temperature on the stability of cocaine in horse blood, sheep vitreous and deer muscle .Forensic Science International , , –.30. , , , , , , , () The effect of sodium fluoride preservative and storage temperature on the stability of 6-acetylmorphine in horse blood, sheep vitreous and deer muscle .Forensic Science International , , –.This work is written by US Government employees and is in the public domain in the US. |