The coagulation system of neonates and children evolves with age, as evidenced by marked physiological differences in the concentration of a majority of hemostatic proteins [1–3]. This concept is known as developmental hemostasis and is well recognized internationally.
Developmental hemostasis has important biological  and clinical implications. Specifically, reference ranges for most hemostatic parameters are analyzer and reagent dependent , and this has critical implications for the definition of healthy children, the diagnosis of disease states and the monitoring of anticoagulant therapy. Both over diagnosis and missed diagnosis are common when age-appropriate, analyzer- and reagent-specific reference ranges are not used. In addition, the impact of developmental hemostasis on the assays used and their underlying principles must be considered in providing the best care for neonates and children.
A number of studies have contributed towards characterizing the specific age-related differences in the hemostatic system, by establishing reference ranges for hemostatic proteins [1–15].
However, it is estimated that only a small number of laboratories worldwide perform clinical testing using adequately established age-, reagent- and analyzer-specific reference ranges. Instead most laboratories use previously published reference ranges. Two of the major factors contributing to this are probably difficulty in accessing blood samples from healthy neonates and children from an ethics standpoint, as well as the cost involved in employing the personnel to collect and analyze the samples. Furthermore, owing to such limitations, currently many hemostatic reference values for preterm infants are lacking, and the ones that have already been reported rely on small study groups [4, 12, 14].
The impact of developmental hemostasis is particularly important in considering the choice of reagents/test kits for specific assays. For example, there are numerous versions of the anti-factor (F)Xa assay available on the market and it is essential to use assays that are physiologically relevant. Hemostatic assays that use additional Antithrombin are not relevant for testing neonatal and pediatric samples, as they overcome the physiological differences and therefore do not provide an accurate, physiological measure of hemostasis [16–19].
Recent evidence demonstrates that the age-specific pattern for the activated partial thromboplastin time (APTT) assay changes depending on the reagent used. Specifically, the APTT reference range decreases with increasing age using the PTT-A reagent (Diagnostica Stago, Asnières sur Seine, France) and increases with age using the Cephascreen reagent from the same manufacturer . Hence, referencing to a published reference range where the only difference is the reagent can lead to mis-diagnosis and should be avoided whenever possible.
The difficulty that laboratories are facing is that the majority of hemostatic reagents currently used in clinical practice do not have published age-appropriate reference ranges available for use. This problem of mis-classification of patients could be solved if age-specific reference ranges were developed prior to reagents being released onto the market.
The outcomes related to patient mis-diagnosis also occur when adult-based reference ranges are used for the diagnosis of pediatric patients. Continuing with the APTT as an example, using adult reference ranges established with the PTT-A reagent  for the diagnosis of 1 to 5 year olds, approximately 30% of children would be classified as abnormal, when in fact they had results within their age-specific reference range using the same reagent and analyzer .
The problem of adequate, validated, reference values is of utmost importance when attempts are made to test and classify premature infants within a normal age-related range of hemostatic parameters.
Misdiagnosis of patients has important consequences for the patient, family, clinician, for the health system as well as for the community in general. For example, investigation of an ‘abnormal’ APTT will usually include a repeat assay, mixing tests, FXII, FXI, FIX, FVIII, von Willebrand factor antigen and collagen binding assay (CBA), with direct unnecessary costs to the health system. Indirect costs may include cancellation of surgery, additional clinical consults, etc. After a mis-diagnosis, the child can be labeled as having a bleeding or clotting disorder, which can in turn have numerous implications for the family for a long period of time.
New assays and devices differences
Recently, whole blood clotting assays are increasingly being used for assessment of clot formation. Numerous assays, utilizing small amounts of whole blood, have been suggested for primary hemostasis evaluation in children and neonates [7,21]. Whole blood-based clotting assays, such as thrombin generation and thromboelastography tests, have also been studied [22–24]. Nevertheless, these assays, mostly reported as single-center studies of cohort groups, still need to be validated as well as age referenced for children, infants and neonates.