Clots in EDTA ‘lavender top' blood samples
In the laboratory, we often receive phone calls from our clinical colleagues with questions about decisions we have made that might affect their patient’s care. For example, enquiries about why we have rejected a sample or why we were unable to provide a particular result due to a pre-analytical or technical error.
In this series of short articles, we want to try and address some of the most common questions we get asked. Our aim is to educate the wider workforce in a way that is simple to understand, about the decisions we make in the laboratory, with the hope that with a greater understanding the number of avoidable errors will be reduced.
In this article, we explore why clots might form in EDTA ‘lavender top’ blood tubes after venepuncture and how, with an understanding of mechanisms involved, these clots can in most cases be avoided.
For healthcare professionals working on the frontline, collecting a blood sample to send off to the laboratory for tests is common practice. Unfortunately, accessing your patient’s records to check for results, only to realise that the sample was not processed because it had clotted prior to analysis is also not too unfamiliar. This means that results are inevitably delayed and usually with negative consequences on your workload, and more importantly, the appropriate and timely care of your patients.
In the haematology laboratory, when we receive requests for investigations, for example the full blood count (FBC), we carry out pre-analytical sample integrity checks, and we often observe that a clot has formed within the sample tube. For haematological investigations that require either the counting or examination of individual blood cells, a blood clot of any size that forms within the tube can cause alterations to the cellular composition of the specimen, making any data collected unreliable. In other words, it achieves very little to process or provide a report on these samples because results produced from a clotted sample will not accurately reflect the in-vivo status of your patient and this is the reason why we would ‘reject’ such specimens.
In order to get a true understanding about the mechanisms involved, we first need to go back to basics with blood tubes. These tubes have different coloured lids for good reasons. It’s important to know that the different colours represent different additives inside the tubes and these colours are denoted by an international standard (ISO 6710:2017). These are designed to preserve and protect the integrity of blood samples pre-, post- and during analysis. It’s important that the appropriate additive is selected when collecting the blood sample for a particular pathology investigation. Selecting the wrong colour top, and therefore the wrong additive could result in clinically significant inaccuracies in the results that are reported, with potentially serious medical consequences.
As haematology scientists, in this article we’re going to explore why these clots can sometimes form in samples collected for haematology investigations, which, for the vast majority of tests, are collected into ‘lavender top’ specimen tubes that contain the anticoagulant Ethylenediaminetetraacetic acid, or ‘EDTA’ for short.
Now let’s take a look at what a clot actually is. To understand this, we’re going to revisit coagulation. Coagulation is a complex aspect of an even more complex process called haemostasis. In normal physiological conditions, it is an inherent property of the blood to prevent excessive blood loss, following damage to blood vessels. In a healthy person, this process kicks into play almost immediately after sustaining damage to the endothelial tissue that lines blood vessels. A very common cause of this damage is venepuncture.
When damage occurs to a blood vessel, it constricts in order to reduce blood flow whilst circulating platelets stick to the site of injury. This event, with the help of a plasma protein called the Von Willebrand Factor, causes the ‘activation’ and ‘aggregation’ of platelets, leading to the formation of a ‘platelet plug’ at the site of damage. The formation of the platelet plug is known as primary haemostasis. Primary haemostasis is swiftly followed by secondary haemostasis - a clot made entirely of platelets is not robust enough to withstand the blood pressure in many blood vessels and so, various other plasma proteins are recruited to strengthen the plug. In a series of interlinked enzyme reactions, these proteins help to make fibrin strands, which forms a strong and supportive mesh around the the platelet plug. This series of events is known as the ‘coagulation cascade’. The moment you break the endothelial lining of your patient’s blood vessel to collect a sample, this cascade of events is initiated and is capable of continuing outside the body within the blood tube after collection. This is why we sometimes observe clots in blood sample tubes.
As we mentioned above, the inside wall of a ‘lavender top’ tube is coated with EDTA which is an anti-coagulant additive that prevents blood clot formation. It also preserves the blood sample to ensure that the constituents to be analysed are not significantly changed prior to the analytical process. With the correct blood sampling procedure, the collected blood is exposed to the EDTA which binds and withholds calcium ions thereby blocking the activation or progression of the coagulation cascade – ultimately inhibiting clot formation. As mentioned earlier, primary haemostasis occurs immediately after venepuncture, so healthcare staff collecting samples must work promptly when collecting samples.
A common cause of clotted EDTA samples is improper mixing of sample tubes after collection. This is often an avoidable event, overcome by inverting the tube 8-10 times immediately after collection to mix the blood thoroughly with the EDTA. These should be gentle inversions, avoiding rigorous shaking. When this is done correctly, the coagulation cascade is blocked, eliminating the possibility of clot formation and these samples then remain stable (suitable for analysis) for up to 24 hours.
What else causes clotting in EDTA tubes and how can they be prevented/avoided? Well, in addition to ‘inadequate’ mixing of the tubes, we have seen simply no mixing of tubes. We have also observed that when syringes are used to collect blood samples into EDTA tubes, they are sometimes over-filled, leaving too little or no air-space that will enable proper mixing during inversions. Inappropriate ‘order of draw’, prolonged venepuncture episodes and mixing-up of sample tube caps (when they are taken off for syringe blood collection method) are also common factors that we have observed to contribute to these events.
It’s for reasons like these we occasionally have to (regrettably) reject specimens, because, like you, we (Biomedical Scientists) as professionals are heavily regulated and have standards of proficiencies that we must abide to – and although we rarely see them, we take great pride in providing the best care we can to our patients, always striving to produce both accurate and precise results. It contradicts our profession's standards of proficiency and professional code of conduct to knowingly report results that are not accurately reflective of the in-vivo activity of our patients.
The take home message is this; in order for us to produce results that provide monitoring and diagnostic value, it is essential that the in-vivo state of our patient remains represented in the blood sample, as at the time of collection from the body. Practitioners tasked with collecting blood samples must be appropriately trained, competent and always follow their local standard operating procedures with respect to venepuncture. Immediate and adequate mixing of the blood sample and the EDTA is critical to avoid the formation of clots.
We invite you to share this article amongst your colleagues and welcome your comments and questions.
By Danny Gaskin (@danieltgaskin) and Ola Yahaya (@molayahaya) - Biomedical Scientists at Furness General Hospital, Haematology and Transfusion laboratory (University Hospitals of Morecambe Bay NHS Foundation Trust)
Moore, G., Knight, G., & Blann, A. Fundamentals of Biomedical Science: Haematology. Oxford: Oxford University Press.