Progress and challenges in the monitoring of hemolysis and clot formation in patients with extracorporeal life support
by Dr Madeleen Bosma
Despite intensive anticoagulation therapy and improved mechanical characteristics of the current systems, hemolysis and clot formation are frequent complications in extracorporeal life support (ECLS/ECMO), which is the standard rescue treatment in patients with severely impaired cardiac and/or pulmonary function. The laboratory aspects of monitoring of hemolysis and hypercoagulation in this patient group are discussed in this short review.
Extracorporeal Life Support (ECLS; also known as extracorporeal membrane oxygenation (ECMO)) is the standard rescue treatment in patients with severely impaired cardiac and/or pulmonary function, including patients severely affected by pandemic influenza or COVID-19. With veno-arterial (VA)-ECLS, both the heart and the lung functions are supported by the ECLS system: blood from a large vein is pumped towards a membrane oxygenator, where gas exchange occurs, and is returned into a large artery. VA-ECLS is also used during and after open heart surgery. In venovenous (VV)-ECLS, the blood is returned into one or two veins and, thus, replaces lung function primarily. Some patients are first supported by VA-ECLS and in later stages by VV-ECLS.
The complication risk with ECLS is high. The patient group is characterized with a high degree of multimorbidity, which enhances susceptibility to complications and mortality. With ECLS, the blood is exposed to various non-biological surfaces as well as (mechanical) shear stress and pressure variations. The coagulation cascade and platelets become activated, which may result in thrombus/clot formation. Clot formation in the ECLS system can lead to increased resistance in the system and eventually to pump failure. Furthermore, thrombi may embolize and, hence, may cause ischemia.
Clot formation leading to obstruction of blood flow, negative pressure and shear stress in the ECLS system are associated with damage to erythrocytes, which ultimately may result in hemolysis [1, 2]. Hemolysis both directly and indirectly (through inflammation) stimulates platelet activation and coagulation, thus further aggravating clot formation [3, 4].
An interesting recent study showed an association between hemolysis and stroke in patients with ECLS . Hemolysis is an independent predictor of mortality . In addition to stimulating a procoagulant state, hemolysis can exert tissue damage through oxidative stress and free hemoglobin may precipitate in the kidneys resulting in renal failure . Additionally, hemolysis can result in NO depletion which is associated with an increased vascular tone, resulting in increased peripheral vascular resistance .
Monitoring hemostasis and clot formation during ECLS
Despite intensive anticoagulation therapy, clot formation in the ECLS system occurs frequently. Monitoring of hemostasis and clot formation is challenging in these patients because of the delicate balance in the risk of bleeding versus the risk of thrombus formation. Exposure to non-biological surfaces, hemodilution, platelet dysfunction, consumption of coagulation factors and platelets, imbalances between coagulation and fibrinolysis contribute to this balance in hypercoagulation versus hypocoagulation . Following the Extracorporeal Life Support Organization (ELSO) guidelines, routine hemostasis status monitoring is performed by the at least daily analysis of fibrinogen, thrombocyte count, activated partial thromboplastin time (PTT), activated clotting time (ACT), anti-Xa activity (aXa), and thromboelastography. The latter is preferred because of the coverage of elements of whole blood that are taken into account (plasma components, fibrinogen, platelets as well as the contribution of heparin and fibrinolysis), but is not always available and is costly. On indication, antithrombin 3 (for acquired heparin resistance/antithrombin deficiency) and heparin-induced thrombotic thrombocytopenia (HITT) tests are performed.
When clot formation is suspected, the system is replaced. Currently, clot formation is monitored by visual inspection of the ECLS circuit (which is only possible in late stages of clot formation and for clot formation in locations that are observable) (Fig. 1), by pressure changes (ΔP) and by a combination of biomarkers. Furthermore, recently several new methods have been presented for clot detection, including CO2 in the exhaust gas , ultrasound dilution technology  and computed tomography . D-dimers are often monitored as a surrogate marker for clot formation. However, D-dimers are often increased in critically ill patients with multi-morbidity because inflammation leads to an increase in D-dimer levels. Therefore, D-dimer levels are often already increased when ECLS is started and are therefore hard to interpret in these patients . Decreases in fibrinogen and platelets may also indicate clot formation. Additionally, hemolysis may be a sign of clot formation, yet will only become observable with extensive clot formation. Hence, there is a clinical need for wellperforming biomarkers for early stages of clot formation. Soluble fibrin (fibrin monomers, represents an earlier phase in clot formation)  and thrombin generation (which may indicate hypercoagulation) may be relevant assays in this context, but require further clinical validation. Hemostasis is a delicate balance, especially in ECLS patients. Furthermore, this patient group frequently presents with multimorbidity and often shows multiorgan failure. Moreover, the group of patients receiving ECLS is a very heterogeneous patient population with the need for personalized precision medicine.
Hemolysis monitoring is important in two ways: (i) as an indicator for extensive clot formation; and (ii) because of the tissue damage that can result from hemolysis. Clot formation in the pumphead is particularly associated with hemolysis [10, 12]. Yet, hemolysis in ECLS is not only due to clot formation – shear stress in the system and pressure variations also contribute. Hemolysis is usually associated with an increase in lactate dehydrogenase (LDH) and a decrease in haptoglobin (which binds and neutralizes free Hb). Therefore, LDH and haptoglobin are used as biomarkers for hemolysis. In this patient group, however, LDH is too non-specific (it is not specific to damage to erythrocytes, it is released with damage to a broad range of cells) and haptoglobin is frequently already decreased or a decrease is masked by an acute phase response. Hence, the best available marker in these patients is free hemoglobin (fHb).
Traditionally, fHb is measured with a laborious spectrometry assay, which is usually only available during office hours. These traditional spectrometry assays are usually developed in-house and vary highly in technical details. Several other assays, including enzymatic and ELISA-based assays, are available for fHb  but are not available on automated platforms. We recently showed that the hemolysis (H)-index of the Roche Cobas platforms, a sample quality control parameter, can also be used as a clinical parameter . The H-index showed good correlation with the conventional fHb assay and showed good analytical performance . Furthermore, the recent release of quality controls for the serum indices have facilitated further implementation of the hemolysis index as a clinical parameter. Thus, the H-index can be used as an easily available low cost clinical parameter for ECLS patients. Boissier et al. have subsequently reported similar results validating our findings . The threshold/ decision limit for the H-index will need to be established in further studies. In our study, a repeatedly observed H-index >20 (equivalent to 20 μmol/L) was associated with mortality .
The H-index is potentially of interest to other patient groups as well, such as patients with the suspicion of a transfusion reaction and in patients with hemolytic anemia. For example, the quickly available and routinely measured H-index may aid in the early diagnosis of severely life-threatening hemolytic anemia, such a Clostridium perfringens infection.
The H-index of every routine clinical chemistry platform can potentially be used, yet not all platforms provide a quantitative H-index but have a qualitative or categorical alternative. Another easily available laboratory parameter that has been suggested as a readout for hemolysis is CO-Hb measured on a point-of-care blood gas analyser ; this may give a quick indication but is an indirect parameter.
In addition to the H-index, the other quality control serum indices of routine clinical chemistry analysers lipemia and icteria may have potential as clinical parameters as well; lipemia, however, may remain unnoticed and is often due to iatrogenic factors .
In summary, there has been substantial progress in the laboratory monitoring of patients with ECLS but significant room for improvement remains. The heterogeneous character of this patient group is an additional challenge, which requires personalized precision medicine. Further insight into the etiology of clot formation and hemolysis, optimization of monitoring strategies and early detection of clot formation in this patient group will be essential. Follow-up studies on this topic are in progress.
Madeleen Bosma PhD
Gelderland Valley Hospital, Willy Brandtlaan 10, 6716 RP Ede,