“Silent killer” – the RAP sheet
With carbon monoxide (CO) being a colourless, odourless and tasteless toxic gas, it is not surprising that the most common analogy used to describe it is the “Silent killer”.
CO is inhaled through the lungs and then directly transferred to the blood stream, where it mainly binds to the oxygen-transporter, haemoglobin, thus preventing the binding of oxygen and its transport to the organs in need, leading to hypoxia [1,2].
However, not all CO binds to haemoglobin: between 10% and 15% of CO entering the blood stream can directly reach other tissues and bind to other proteins or compounds, leading to direct cellular toxicity [3,4].
Depending on the amount and duration of exposure to CO, severe short- or long-term adverse health effects involving the respiratory, cardio-circulatory or neurological systems can occur and, in worst cases, lead to death.
The importance of CO poisonings on a global scale is confirmed by the addition of CO estimates in the most recent report from the Global Burden of Disease (GBD) in 2017, which reported 35000 deaths and 1462.4 disability-adjusted life years associated with CO exposure for that year [5].
Despite mortality having decreased in the past 25 years, the incidence of CO poisoning remains stable and the issues that need to be addressed are the same: prevention, correct diagnosis and treatment [6].
Prevention is achieved through public health strategies that include awareness campaigns, prevention plans and research – the CO Research Trust is a very good example on how to accomplish some of those tasks.
Concerning the diagnostic process, there are several issues that need to be addressed. One of the major ones is the non-specificity of the reported symptoms (e.g. dizziness, nausea, headaches, etc.), which often lead to misdiagnoses by both the patient and the clinician. Due to the lack of suspicion, no test to measure the CO levels is performed, generating a high number of undiagnosed cases [7–9].
Another issue is the increased number of cases describing inconsistencies between reported symptoms and measured CO levels [10]. While it is possible that a part of these differences are related to distinct inter-personal characteristics (lung volume, ventilation rate, age, various pre-existing morbidities) [11,12], the accuracy of the methods used for CO measurement is also a factor that needs to be considered.
The choice of the measurement technique employed depends on the biomarker selected for the measurement. For CO, this choice has fallen onto carboxyhaemoglobin (COHb).
However, since COHb is not solely responsible for the CO-related toxicity, quantifying the total amount of CO in blood might help explain the inconsistencies in physiological and neurological outcomes associated with CO and reduce misdiagnoses.
Notice of allegations
The main objective of this project, which was funded by the CO Research Trust, was to find a more accurate and robust analytical method for CO poisoning determination, capable of measuring the total amount of CO in blood, as alternative to the current spectrophotometric COHb determination, to help improve diagnostic efficiency.
After comparison of the newly developed with the standard method, another part of the project was dedicated to determining potential sources of measurement error and quantifying the impact of these errors, such as storage parameters, on changes in the CO/COHb concentrations over time.
The investigative approach
Based on a thorough literature review to generate an overview of the current state-of-the-art regarding sample storage, preparation and measurement techniques in CO poisonings, the most appropriate measurement technique considered to have the potential of measuring CO with higher accuracy was an approach based on gas-chromatography coupled to mass spectrometry (GC-MS).
In very basic terms, GC-MS is an analytical method capable of separating the compounds present in a mixture and identifying as well as quantifying them based on their different physical and chemical properties (e.g. mass, volatility, structural formula).
The advantages of using a GC-MS based approach are that CO will be measured more precisely and accurately, with the method being also more specific and less subject to interferences that might be in the sample due to poor sampling conditions or the sample quality itself - blood from burn victims might be coagulated and thus cause interferences with the traditional gas analysis.
Furthermore, GC-MS offers the opportunity to measure the total amount of CO in blood (TBCO), which includes both the CO bound to hemoglobin as well as the free CO in the blood that directly reaches the tissues. The main disadvantage is that GC-MS methods require an instrument that is more expensive and higher maintenance compared to a spectrophotometric method that uses a gas analyzer, which is a common instrument found in most EDs and clinical labs.
In this project, samples were collected from both clinical and forensic cases. For the clinical cases, samples from before and after CO exposure were obtained, thus giving a unique opportunity to directly measure the increases in both compared biomarkers, COHb and TBCO.
For the forensic cases, only postmortem samples were obtained, which included non-CO related deaths as a control group as well as different degrees of CO-related deaths. In a different, in-vitro study, a variety of storage parameters were investigated.
Did we find the culprit?
The measurement of COHb via spectrophotometry, such as a blood gas analysis or UV-spectrophotometer, is currently the method of choice for CO exposure determination.
However, several analytical issues were identified with this method, including the dependence on the samples’ quality and also low accuracy especially for lower CO exposure levels, which make the analysis in many cases either very difficult to impossible or with falsely elevated or decreased results – this can be relevant especially in non-suspicious clinical cases or in forensic circumstances.
The search for an alternative biomarker found in the measurement of TBCO via GC-MS a very promising candidate. Results from the clinical samples showed that TBCO resulted in generally higher results compared to COHb, which supports the hypothesis of the important role of measuring the total blood CO as opposed to just the CO bound to haemoglobin.
Furthermore, comparison of the storage parameters showed that TBCO was more stable over time and less affected by changes in storage temperature or the sampling method than COHb.
Outlook
One shortcoming of this project that needs to be addressed was the limited number of cases analysed in both clinical and forensic settings. To strengthen these results from a statistical perspective, a higher number of samples in each group need to be included.
In addition, there is not enough data on the interpretation of TBCO values – the analysis of more samples will allow the generation of a reference table that associoates TBCO values to the degree of poisoning and the symptomatology.
Nevertheless, this work established a foundation and provided some valuable tools to help future researchers to continue working towards closing the knowledge gap on CO poisoning diagnoses.
With thanks to Dr Stefania Oliverio, Forensic Toxicologist at the National Health Laboratory in Luxemburg, department of forensic medicine.
Dr Oliverio will be presenting the findings of her project during our February lecture. The lecture will take place online on Mon 20th Feb between 2-3pm - you can sign up to attend here.
References
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10. Raub JA, Mathieu-Nolf M, Hampson NB, Thom SR. Carbon monoxide poisoning - A public health perspective. Toxicology. 2000;145:1–14.
11. Bleecker ML. Carbon monoxide intoxication. Handb Clin Neurol. 3rd ed. Oxford, UK: Elsevier B.V.; 2015. p. 191–203.
12. Penney DG. Carbon Monoxide Poisoning. 1st ed. Group T& F, editor. Boca Raton: CRC Press; 2007.