How to take the perfect neonatal clotting sample

Mia Kahvo, Portia Cartwright, Beth Osmond

Introduction

The coagulation screen is a useful diagnostic test, however it is also one that can not only be difficult to interpret but also to perform. The prompt recognition and investigation of bleeding disorders is vital to allow correct diagnosis and appropriate management of any bleeding disorder. Central to this is taking a high-quality blood sample, often venous, which can be challenging, particularly in unwell neonates. A spot audit in our neonatal intensive care unit found that 30% of all coagulation samples taken in a 1-month period were ‘rejected’. Reasons ranged from the sample bottle being underfilled or heparin contamination to clotted specimens (most common), and all resulted in repeat samples needing to be sent. As we have evidence to suggest that repeated painful procedures in newborns are linked to physiologic instability and altered stress responses(1), as well as the risk of iatrogenic anaemia(2) when repeating ‘failed’ samples, how can we ensure that the samples we take are of good enough quality to be processed in the lab?

Here’s some questions for you to ponder (answers at the end of the post)

What factors influence whether a lab test will produce accurate results?

It is important as a front-line clinician to have a basic understanding of factors which can influence a clinical test giving an inaccurate result. Do not make the assumption that because a clotting sample is reported by the lab as “clotted” that the infant has a normal clotting ability. Accurate test results and avoidance of repeated sampling are dependent on a number of variables:

1. Pre-analytical variables affect the sampling process and form the first part of the sample journey. Errors here, such as incorrect test tube selection or poor sample handling, can therefore have a negative effect on all other processes downstream and lead to inaccurate results.
2. Analytical variables involve the laboratory testing procedures and can be affected by inadequate quality control of machinery and assays used for testing.    
3. Post-analytical variables occur after the result is generated, such as incorrect data transmission (for example hearing information relayed verbally, incorrectly).

Steps involved in the pre-analytical stage are therefore the easiest for us to influence to ensure we send a good quality sample to the lab.

When we consider optimising pre-analytical variables, it is useful to have a basic understanding of coagulation. The haemostatic process can broadly be divided into 3 phases:

1. Formation of a platelet plug following injury to vessel subendothelium
2. Formation of a cross-linked fibrin clot (the coagulation cascade- Figure 1)
3. Removal of formed clots after haemostasis has occurred (fibrinolysis)

 Clotting is initiated as soon as there has been injury to the vessel wall- circulating platelets bind to exposed collagen creating a ‘platelet plug’, strengthened by von Willebrand Factor. What then follows is an immediate and complex interaction between platelets, circulating cells and plasma proteins leading to a local response at the site of injury and resulting finally in tissue repair and thrombosis formation.

For the purposes of good blood sampling, it is important to consider the effects that tissue factor and ionised calcium have on the coagulation cascade (Figure 1). Tissue factor is the primary initiator of the cascade(4). Following vessel injury, it forms a complex with Factor VIIa, marking the first step in the extrinsic pathway (the dominant pathway in coagulation). It can also be activated by tissue damage outside of the blood vessel, sepsis, hypoxia and inflammation.

Calcium plays an equally crucial role by aiding platelet adhesion as well as acting as a co-factor in several enzymatic processes(5). From Figure 1, we can see that without calcium, the coagulation process can be severely impaired, and this in fact forms the basis of how whole blood is altered in the coagulation test tube. These test tubes contain 3.2% sodium citrate which acts a calcium inhibitor, therefore arresting the coagulation process. Once in the lab, the blood sample is then recalcified to re-start coagulation. For this process to function properly however, there has to be a specific ratio of blood to citrate (9:1) and this is the reason why coagulation bottles need to be filled more precisely than other samples.

Applying these principles to our blood taking process:

When it comes to clotting, the blood sampling technique is very important- the more traumatic the process of taking blood, such as squeezing a limb, then the more time there is for the cascade to be initiated resulting in partially clotted blood filling the tube. Avoid collecting blood in a syringe and instead aim to fill the test tube directly.

While not generally recommended, it is sometimes necessary to collect blood from a capillary sample. This is done in the knowledge that clotting times may be inaccurate and so should be interpreted with caution. However, to reduce the risks, there are a number of steps we can take:

• Ensure the heel is warmed prior to sampling
• Only sample free flowing blood and avoid ‘squeezing’ the heel, as this increases release of tissue factor and speeds up the clotting process further
• Wipe away the first drop of blood after ‘pricking’ the infant’s heel, as this often contains excess tissue factor or plasma that can dilute the sample
• Do not collect blood in a capillary tube and then transfer to the sample bottle- the capillary tube contains heparin and so will affect your results!

This figure shows the blood sampling and handling process, and how errors introduced in the pre-analytical phase can have a negative impact on the end result.

Additional principles to bear in mind from those listed above are the order that test bottles are filled and the site of blood sampling. Both EDTA and lithium-heparin are powerful anticoagulants. Not filling the coagulation bottle first risks cross-contamination and therefore false clotting times once the sample in processed in the lab. Similar principles apply if blood is being sampled from central lines which are often heparinised. Therefore, always fill the coagulation bottle first and if sampling blood from a central line, ensure that an appropriate amount of blood is first discarded before the test tube is filled.

Conclusion

Monitoring coagulation is important in the neonatal unit and can impact upon clinical management. While complex, understanding the basics of the coagulation cascade can provide a number of clues as to how to best optimise pre-analytical variables and ensure accurate coagulation times in the lab. This ranges from the speed and technique of sampling (to limit time for cascade initiation), to the amount of blood we collect (ensuring an adequate ratio of sodium citrate and blood to inactivate calcium). 

How to improve your clotting sample success rate:

1	Ensure the sample bottle is filled exactly to the bottle ‘fill’ line.
2	Take time selecting an appropriate vein for the blood draw. If taking blood from a capillary sample, avoid squeezing the heel and wipe away the first drop of blood. Interpret results with caution.
3	Once collected, gently invert the blood bottle 3-6 times to ensure adequate mixing of blood and sodium citrate. Avoid shaking the tube as this can lead to in vitro haemolysis or spurious tissue factor activation resulting in false shortening of clotting times!
4	Sample blood in the coagulation tube first to avoid cross-contamination with anticoagulant factors present in EDTA and lithium-heparin tubes.
5	Ensure the sample reaches the lab for processing within 4 hours.

References:

1.            MEDICINE COFANaSOAAP. Prevention and Management of Procedural Pain in the Neonate: An Update. Pediatrics. 2016;137(2):e20154271.

2.            Widness JA. Pathophysiology of Anemia During the Neonatal Period, Including Anemia of Prematurity. Neoreviews. 2008;9(11):e520.

3.            Gleason CA, Juul SE. Avery’s diseases of the newborn. Tenth edition. ed. Philadelphia, PA: Elsevier; 2018. xxvii, 1627 pages p.

4.            Mackman N. Role of tissue factor in hemostasis, thrombosis, and vascular development. Arterioscler Thromb Vasc Biol. 2004;24(6):1015-22.

5.            Mikaelsson M. The Role of Calcium in Coagulation and Anticoagulation. In: Sibinga C, Das P, Mannucci P, editors. Coagulation and Blood Transfusion Developments in Hematology and Immunology. 26. Boston, MA: Springer; 1991.