Pediatric Fibrinogen PART I—Pitfalls in Fibrinogen Evaluation and Use of Fibrinogen Replacement Products in Children

Fibrinogen is a key coagulation protein, playing a critical role in hemostasis. It is the first factor to decrease to critical levels during bleeding. Hypofibrinogenemia is an important risk factor for bleeding in clinical settings, including pediatric surgery. Yet, the optimal measurement of fibrinogen levels is subject to debate, as is the critical threshold for intervention. Fibrinogen replacement may be provided by cryoprecipitate and fibrinogen concentrate. Whilst both products contain fibrinogen, they are not equivalent, each has its own advantages and disadvantages, especially for pediatric use. Unfortunately, medical literature to support fibrinogen replacement in children is limited. In this article we review the current diagnostic tools to measure fibrinogen, with respect to their use in the pediatric critical care setting. Secondly, we evaluate the different fibrinogen replacement therapies, focusing on cryoprecipitate and fibrinogen concentrate and examine their individual product characteristics, associated risks and benefits, different dosing strategies and specific pitfalls for use in children. We summarize by highlighting current knowledge gaps and areas for future research.


INTRODUCTION
Fibrinogen (Factor I) is a key coagulation protein and plays a critical role in all aspects of normal hemostasis: from platelet aggregation, to clot formation, and fibrinolysis (1,2). During major surgical bleeding, fibrinogen is the first hemostatic factor to decrease to critical levels (3). Fibrinogen has been identified as an important risk factor for bleeding in children undergoing surgery (4)(5)(6).
As an acute phase reactant, fibrinogen plays a pivotal role in tissue repair and maintaining hemostasis during tissue injury and inflammation (7). Levels are often elevated in children in the context of fever, sepsis and critical illness (8)(9)(10). However, if hypofibrinogenemia is present in critically ill children with sepsis, it is associated with increased in-hospital mortality (8,9).
See "Pediatric Fibrinogen PART II-Overview of Indications for Fibrinogen Use in Critically Ill Children", for further information about the clinical indications for fibrinogen supplementation in children.

Fibrinogen Evaluation and Specific Pitfalls in the Pediatric Setting
The gold-standard for fibrinogen quantification is the clot-based Clauss fibrinogen assay. The Clauss-assay is a functional test that measures the time taken for plasma to clot in a high concentration of thrombin, with comparison against a reference plasma calibration curve (11). Patient factors that can impact the accuracy of the test include the presence of heparin and fibrinogen-degradation products (12). Similarly photo-optical clot detection systems for measuring fibrinogen may be affected by lipemia or hyperbilirubinemia (11). Hydroxyethyl starch (HES), a plasma volume expander used in some pediatric perioperative settings (13), can impair fibrin polymerization (13,14) causing falsely reduced fibrinogen levels (6,14). Additionally, direct thrombin inhibitors such as bivalirudin and argatroban can inhibit the thrombin used in the Clauss-assay causing falsely reduced levels (15).
Fibrinogen may also be evaluated by viscoelastic testing. The two most commonly used and evaluated in clinical trials in children are thromboelastography (TEG R ) and thromboelastometry (ROTEM R ). A qualitative indication of fibrinogen is obtained by inhibiting the platelet contribution to clot formation using cytochalasin D for ROTEM-FIBTEM and abciximab for TEG functional fibrinogen (TEG-FF) assay (16).
Whilst both TEG and ROTEM are based on similar methodologies, there are significant differences in operating characteristics, activators used, reporting nomenclature, and reference ranges. Parameters reported by each of the devices are not equivalent or interchangeable (16). One such example, is the measurement of hyperfibrinolysis, the TEG lysis index is measured 30 min after the maximum amplitude (MA), whereas the ROTEM lysis index is measured 30 min after the clotting time (17).
Viscoelastic measures of fibrinogen have benefits over the Clauss assay, since they are point-of-care test with quick turnaround times (16). Since they are performed using whole blood, they provide a global and visual hemostatic evaluation of hemostasis, from clot initiation and kinetics, through to clot degradation and fibrinolysis.
In the pediatric setting the usefulness and uptake of viscoelastic testing remains limited. There is a lack of standardization, with a scarcity of age-dependent reference ranges for the different reagents used with each viscoelastic method (18,19). Studies are needed to define clear thresholds and targets for treatment in neonates and children (17). The maximum clot strength thresholds (Maximum Clot Firmness [MCF] for ROTEM or MA for TEG) that indicate the need for fibrinogen supplementation are debated. In addition, validated treatment algorithms using viscoelastic testing and fibrinogen supplementation are needed for the different neonatal and pediatric contexts (17).
Viscoelastic testing in infants and neonates may also be limited by the blood sample requirements; ROTEM requires at least 1.8 mL of whole blood and TEG 2.7 mL (20) although newer viscoelastic devices have smaller minimum sample requirements (17). Another limitation is that viscoelastic tests are unable to detect congenital bleeding disorders, such as platelet function disorders or von Willebrand disease (17).
Other techniques for measuring fibrinogen include Prothrombin-time (PT) derived tests, immunohematological assays (11), the dry-hematology method (21), and thrombin generation assays. These tests are not widely used or only available in research settings (22).
The fibrinogen level obtained will vary depending on the fibrinogen assay used (23). In adults, fibrinogen levels using the Clauss assay are typically reported between 150 and 450 mg/dL, but physiological variation is commonly seen. In children, age-related reference ranges are required for fibrinogen quantification, because of the age-dependency (the so called developmental hemostasis) in hemostatic proteins (24). The lowest fibrinogen levels are reported in fetuses and preterm neonates (25)(26)(27), and infants (28,29). Similarly, there are agedependent differences seen in viscoelastic testing results (19,(30)(31)(32). Pediatric age-specific reference ranges have been described for ROTEM parameters across pediatric age groups, including FIBTEM (31,33) and for the TEG (19,34).
Children also have qualitative differences in both fibrinogen and fibrinolysis (35). Neonates have a "fetal" form of fibrinogen (35), an altered fibrin network and clot structure (36), with an overall reduction in fibrinolytic activity (37), and often physiologically elevated D-dimers in the first 72 h of life (38).
The sensitivity of each fibrinogen assay refers to the lowest detectable and quantifiable amount of fibrinogen. The lowest reportable fibrinogen level will vary between individual laboratories but will be reported as less than a certain threshold, e.g., <50 mg/dL. In neonates, this can be rather imprecise, because physiological values can be this low.
In summary, fibrinogen reference ranges vary depending on the test, the analyzer and reagents used, in addition to the age of the child, therefore it is important that local, assay-specific and age-specific reference ranges are developed. Utilization of adult reference intervals, particularly in the neonates has the potential to lead to overtreatment. A normal physiological (low) fibrinogen level in a non-bleeding, preterm neonate, for example, does not need correction.

FIBRINOGEN SUPPLEMENTATION
Cryoprecipitate and fibrinogen concentrate both effectively restore fibrinogen levels (39,40), and are used in children to provide fibrinogen replacement during active bleeding or as prophylaxis to prevent bleeding (41,42). Internationally there is variability in practice regarding the favored fibrinogen replacement product, reflecting local legislation, licensing and product availability.

Cryoprecipitate
In the United States (US), Canada, United Kingdom (UK), Australia and New Zealand, cryoprecipitate is the main component available for treatment of acquired hypofibrinogenemia since fibrinogen concentrate is only licensed for the treatment of congenital fibrinogen deficiency (43).

Fibrinogen Concentrate
In contrast, many European countries favor fibrinogen concentrate over cryoprecipitate for all therapeutic uses due to its superior pathogen safety profile and cryoprecipitate has been withdrawn due to safety concerns, principally transfusion transmitted infection (TTI) and prions. Fibrinogen concentrate in Europe is licensed for treatment of both congenital and acquired hypofibrinogenemia (43).
Whilst cryoprecipitate and fibrinogen concentrate are both plasma-derived products, there are considerable differences between the two products (see Table 1).

Plasma Transfusion
Plasma is the liquid component of blood that contains coagulation factors and coagulation inhibitors. Plasma products available include plasma frozen within 6-8 h of collection (FFP), plasma frozen within 24 h of collection, thawed plasma, liquid plasma and pathogen-inactivated plasma (44)(45)(46). Freeze-dried (lyophilized) or spray-dried plasma is largely restricted to military and research settings (47).
In the pediatric setting however, none of these plasma products, are suitable as fibrinogen replacement products since fibrinogen concentrations in plasma are very low and can vary considerably (100-500 mg/dL) (48,49). Large volumes of plasma are required to replenish a low or falling fibrinogen level (43), placing a child at significant risk of transfusion-associated circulatory overload (TACO).
In summary, cryoprecipitate and fibrinogen concentrate are superior to plasma as fibrinogen replacement products since they are concentrated products. We therefore will focus this review on these two fibrinogen products.

PRODUCT CHARACTERISTICS Product Characteristics of Cryoprecipitate
Cryoprecipitate is the unpurified, cold-insoluble protein or "cryoglobulin" proportion derived from plasma. Cryoprecipitate may be manufactured as single apheresis units or pooled from multiple donors with varying volume size. It is stored  as a frozen product and inventoried by blood group. It contains fibrinogen in addition to coagulation factors VIII (FVIII), von Willebrand factor (vWF), factor XIII (FXIII), as well as fibronectin and platelet microparticles (45). Cryoprecipitate was originally used therapeutically in the treatment of hemophilia A, and then von Willebrand disease (vWD), congenital fibrinogen and FXIII deficiency (50). Today cryoprecipitate is predominantly utilized to replace fibrinogen in acquired hypofibrinogenemia. Internationally, specific product requirements for the manufacture of cryoprecipitate vary between countries with respect to the minimum fibrinogen, FVIII and vWF concentrations (44) (see Table 2). Whilst minimum fibrinogen content per unit is usually specified by standards (e.g., >140 mg/unit) (45,46), the actual concentration can vary considerably (300-3,000 mg/dL), due to differences in blood donor fibrinogen levels, varying manufacturing processes and different unit volumes (51).
Advantages and disadvantages of cryoprecipitate in comparison with fibrinogen concentrate are described in Table 1.
One potential clinical advantage of cryoprecipitate is the contribution of FXIII in the treatment of bleeding, especially in the surgical setting (52)(53)(54). In-vitro studies have shown that cryoprecipitate reverses fibrinolysis better than fibrinogen concentrate (55). A second consideration is cost: when evaluated per gram of fibrinogen, cryoprecipitate is cheaper (56,57), even when economic analysis has considered preparation and wastage costs (58). However, this advantage may not be as notable with pathogen-reduced cryoprecipitate, since pathogen-inactivation comes with additional costs.
An important downside of cryoprecipitate is the risk of pathogen transmission, even with appropriate blood donor and donation screening (59). Cryoprecipitate made from pathogenreduced plasma can reduce this risk (60)(61)(62) and this is the preferred product for the treatment of congenital bleeding disorder in resource-limited settings (60,61). Unfortunately, pathogen-inactivation results in reduced fibrinogen levels (65-84%) of normal plasma (62). To overcome this, it may be possible to increase the number of donors contributing to the plasma pool or increase the transfusion volume (63, 64).
Cryoprecipitate has been implicated in a number of transfusion-related adverse events including allergic reactions, febrile, non-hemolytic transfusion reactions (65,66) as well as transfusion-associated acute lung injury (TRALI) (67,68) and TTI (59,69). There is also evidence that adverse transfusion reactions occur more frequently in children compared with adult (66,70). Any patient receiving cryoprecipitate must be monitored for a transfusion reaction and any incidents should be reported to the local hemovigilance reporting system. To maintain traceability, the blood compatibility report should be maintained in the child's medical record.
Cryoprecipitate is less suited to the acute pediatric critical care setting since it requires thawing prior to administration, and once thawed, it has a shelf life of 4 h, after which it must be discarded (46). To overcome this, it would be ideal to keep "ready for-use" units of unthawed cryoprecipitate. In-vitro studies evaluating thawed pathogen-reduced cryoprecipitate, held at room temperature for 5 days have demonstrated its ability to restore fibrinogen levels and clot strength (FIBTEM) (63). However, in the pediatric setting di (2-ethylhexyl)phthalate (DEHP) toxicity should be considered (71). DEHP is a chemical added to plastics to make them more flexible and is commonly used in blood bags (72). DEHP levels increase in concentration as blood storage duration is increased (71). Toxic and carcinogenic effects are seen in animal studies, but the evidence for toxicity in humans has not been established, but remains a concern in neonates (72).

Product Characteristics of Fibrinogen Concentrate
Fibrinogen concentrate is a purified, virus-inactivated, lyophilized concentrate derived from pooled human plasma. It comes as a powder that can be reconstituted with sterile water (within 5-10 min) to deliver a reliable and standardized fibrinogen content (73,74). In children with hypofibrinogenemia, fibrinogen concentrate has a rapid onset of action and is effective at increasing fibrinogen levels (39,40).
An overview of the advantages and disadvantages of fibrinogen concentrate compared with cryoprecipitate are described in detail in Table 1. An advantage of fibrinogen concentrate for use in neonates and critically ill children is its relatively small infusion volume. Doses of fibrinogen concentrate are going to be in the order of 1-3.5 mL/kg compared with 5-10 mL/kg for cryoprecipitate. Fibrinogen may be able to be administered quicker than cryoprecipitate, depending on where it is stored in the hospital, since it only requires refrigeration for storage. Fibrinogen concentrate also has a superior pathogen safety profile and low rates of adverse events. A pharmacosurveillance study of 27 years of Haemocomplettan P R /RiaSTAP R (CSL Behring) use with literature review, reported an excellent safety profile (85). Each fibrinogen concentrate administration be monitored for side effects and any reactions or adverse events be reported to the pharmacovigilance system. Each administration and the batch number must be recorded in the patient record to maintain traceability.
A relative disadvantage of use in neonates is that the dose required for treatment is much less than the smallest formulation of fibrinogen concentrate available (1,000 mg), leading to product wastage and higher costs. For example, a dose of 30 mg/kg fibrinogen concentrate to treat hypofibrinogenemia in a 5 kg infant only equals 150 mg.
In summary, cryoprecipitate is a rich source of fibrinogen and includes other coagulation factors important for hemostasis. Cryoprecipitate is widely used in countries where fibrinogen concentrate is not licensed for use in acquired hypofibrinogenemia, and in resource-limited settings as therapy for congenital hypofibrinogenemia, although pathogen-inactivated cryoprecipitate is favored. Disadvantages are the large inter-unit variability of fibrinogen in cryoprecipitate, the increased risk of transfusion reactions and infectious transmission, in addition to the logistical and blood banking requirements.
Plasma-derived fibrinogen concentrates are able to rapidly and effectively restore and maintain serum fibrinogen concentrations. They are used for treatment of congenital and acquired hypofibrinogenemia. They have an excellent safety profile and are amenable to near-patient storage, which may reduce the time to administration. They come with a higher cost and generally do not contain additional hemostatic factors that may be important for general hemostasis.

Dosing of Cryoprecipitate
A standard treatment dose of cryoprecipitate in adults is 10 units or two pools (where one pool is made from five units), leading Takes into account the desired fibrinogen increment (mg/dL), average fibrinogen content per unit of 250 mg and plasma volume of the child (9). Plasma and blood volumes vary in children.
Blood volumes in children may be estimated using the formula 70 mL/kg, but this will underestimate the blood volume in a neonate and overestimate the blood volume in an adolescent or overweight child (10,11) Blood to an increase of 100 mg/dL fibrinogen (86). In children, dosing for both prophylactic and therapeutic cryoprecipitate indications should be calculated at least taking into account the child's weight (87). Most pediatric transfusion guidelines dose cryoprecipitate based on the child's weight as a single variable (88,89). Many advise doses of 5 and 10 mL/kg (88)(89)(90)(91), with exceptions of 20 mL/kg for the treatment of congenital fibrinogen deficiency with pathogen-reduced cryoprecipitate (64) (see Table 4).
However, more complex formulas will take into account a child's baseline fibrinogen level, the desired fibrinogen level, the average fibrinogen content of the local cryoprecipitate unit and a child's plasma volume (92) (see Table 4).
In general, blood volumes in children may be estimated using the formula 70 mL/kg (93,94). However, this equation underestimates the blood volume for a neonate, since the estimated blood volume (EBV) decreases with age, from around 90-100 mL/kg in preterm infants to ∼80 mL/kg in term infants (93) and overestimates the blood volume in obese, post-pubertal adolescents (∼60-70 mL/kg). None of these calculations corrects for ethnic differences and social factors such as malnutrition or obesity which may be important (94).
Dosing of cryoprecipitate in children should also consider the fibrinogen content of local products, particularly with pathogenreduced cryoprecipitate (92,95,96).
The half-life of fibrinogen is relatively long (3-4 days) (64), and therefore, usually one dose is sufficient for prophylactic indications, however in the presence of active bleeding with ongoing loss or a poor fibrinogen increment, additional doses may be required.

Dosing of Fibrinogen Concentrate
Fibrinogen concentrate is dosed by most clinical guidelines in mg/kg. But here also, more complex formulas are seen, taking target and measured fibrinogen levels into the equation (see Table 4).
For each of the other available fibrinogen concentrates on the market, there are different dosing recommendations for children, and it is therefore recommended to consult the individual product information for specific dosing advice (see Table 3).
In summary, the dosing of cryoprecipitate is influenced by both donor and product variables. The dosing of both cryoprecipitate and fibrinogen concentrate in children should be calculated, taking into consideration the baseline fibrinogen, the child's body weight, the presence of active bleeding and ongoing loss. It is advisable, that after fibrinogen supplementation, both the clinical response to treatment and fibrinogen levels are re-evaluated to assess for any additional requirements.

CURRENT KNOWLEDGE GAPS, CONTROVERSIES, AND AREAS FOR RESEARCH
The decision to supplement fibrinogen firstly relies on adequate measurement of fibrinogen. When evaluating fibrinogen levels and function in the critically ill child, it is important to consider the accuracy of the result, the clinical context, the specific reference ranges and each test's limitations.
When fibrinogen replacement is indicated in critically ill children, there remain many uncertainties regarding the best choice of fibrinogen replacement, the optimal dose, in addition to the target or desired fibrinogen level. Each product has its own set of limitations and benefits specific to children.
The following questions, may be proposed as potential areas for research in neonates, including those preterm, infants, children, and adolescents. In the area of laboratory testing: • Can we develop small-volume tests for measuring fibrinogen in neonates and critically ill children?
• How do different viscoelastic measures of fibrinogen function compare with clot based Clauss fibrinogen assays in children?
Fibrinogen and bleeding • How do age-dependent coagulation differences influence bleeding in neonates and children? • In particular, what effect does the presence of fetal fibrinogen and altered fibrin clot structure have on fibrinogen function in preterm and term neonates?
Fibrinogen replacement products • What is the optimal fibrinogen replacement component in children?
• What role does the additional FXIII, vWF, and FVIII in cryoprecipitate play in treating and preventing bleeding in children with a low fibrinogen? • How do fibrinogen concentrate products that contain FXIII compare with those that do not? • What is the optimal formula to dose fibrinogen in children?
• What is the optimal dose and timing in children of cryoprecipitate and fibrinogen concentrate • for the prevention of bleeding?
• for the treatment of active bleeding?
Adverse events: • How do we capture and report adverse events related to fibrinogen supplementation in children?

CONCLUSIONS
Hypofibrinogenemia is increasingly recognized as an important risk factor for bleeding and there has been an increasing focus on the fibrinogen supplementation by clinicians and clinical guidelines. Yet, there are many unknowns. The decision to supplement fibrinogen firstly relies on adequate measurement of fibrinogen and there are many pitfalls around the optimal fibrinogen measurement in children. Cryoprecipitate and fibrinogen concentrate both effectively restore fibrinogen levels, but each product has its own set of advantages and constraints specific to use in children. Fibrinogen concentrate is an attractive alternative to cryoprecipitate, offering a superior safety profile, with apparent efficacy, but not every fibrinogen concentrate product is equivalent. Further randomized controlled evidence is required to support decisionmaking regarding fibrinogen supplementation in children, including those who are critically ill.

AUTHOR CONTRIBUTIONS
GC literature review, writing, and editing article. EH reviewing content and editing article. All authors contributed to the article and approved the submitted version.