Developmental hemostasis: recommendations for laboratories reporting pediatric samples

Authors

  • V. IGNJATOVIC,

    1. Murdoch Childrens Research Institute, Royal Childrens Hospital, Parkville, Victoria
    2. Department of Paediatrics, Royal Childrens Hospital, The University of Melbourne, Parkville, Victoria, Australia
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  • G. KENET,

    1. Thrombosis Unit, National Hemophilia Center, Sheba Medical Center, Tel-Hashomer
    2. The Sackler Medical School, Tel Aviv University, Tel Aviv, Israel; and
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  • P. MONAGLE,

    1. Murdoch Childrens Research Institute, Royal Childrens Hospital, Parkville, Victoria
    2. Department of Paediatrics, Royal Childrens Hospital, The University of Melbourne, Parkville, Victoria, Australia
    3. Department of Clinical Haematology, Royal Childrens Hospital, Parkville, Victoria, Australia
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  • On behalf of the perinatal and paediatric haemostasis subcommittee of the scientific and standardization committee of the international society on thrombosis and haemostasis


Vera Ignjatovic, Haematology Research Laboratory, Murdoch Childrens Research Institute, Flemington Road, Parkville, Vic. 3052, Australia.
Tel.: +61 3 99366520.
E-mail: verai@unimelb.edu.au

Introduction

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 [4] and clinical implications. Specifically, reference ranges for most hemostatic parameters are analyzer and reagent dependent [4], 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].

Reagent differences

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 [20]. 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.

Age differences

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 [11] 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 [11].

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.

Recommendations

Diagnostic laboratories processing pediatric samples should use age, analyzer and reagent appropriate reference ranges. Furthermore, where possible, tests/reagents that are as close as possible to physiologically relevant (i.e. no additional AT) should also be used. These two factors are essential for making a patient appropriate and physiologically relevant diagnosis. In developing age-appropriate reference ranges, laboratories should endeavor to standardize age groups (i.e. neonates, 1 month to 1 year, 1- to 5-, 6- to 10-, 11- to 16-year-old children), which have been reported to reflect statistically different groupings [1–3]. Further studies will be required to better delineate changes in the < 1-year-old age group. In the absence of being able to generate their own reference ranges, laboratories should only use published ranges that were established using the identical analyzer and reagent system to that used in their laboratory. This alternative is based on the fact that results can be compared across laboratories, providing the population, reagents and analyser is identical. This is because population-specific differences have been demonstrated previously [25], reagent-specific differences have been demonstrated previously [11,19] and analyzer-specific differences have been previously demonstrated [25]. If published data for the analyzer/reagent system in use at a laboratory are not available, then the laboratory must be very cautious about performing and interpreting coagulation studies in children, and should consider referral of samples to a laboratory that can provide age-appropriate reference ranges.

With recently advanced neonatal care and increased survival of very young, low-birth-weight and sometimes extremely sick premature infants, the issue of validated reference values for all hemostatic proteins and assays in premature neonates deserves urgent attention. New devices and techniques for hemostasis assessment should be approached with structured, age-related research in order to define specific age-related reference values.

Conclusions

In order to provide the best possible standard of care for infants and children, hemostasis laboratories must use population-, reagent- and analyzer-specific reference ranges.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

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