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Background: The detection of deficiencies in B 12 and folate children is important. However, despite the availability of various markers to assess B 12 and folate metabolism, there are limited studies describing the reference intervals (RIs) and changes during growth and development for these markers in healthy children., Methods: Using samples collected from 378 children aged 30 days-< 18 years, we derived continuous RIs for holotranscobalamin, homocysteine and red cell folate., Results: The lower RI for holotranscobalamin was lowest at birth, rising during early childhood and then declining following ages 4-6 years whereas red cell folate was highest early in life and then declined steadily towards adulthood. Total homocysteine, reflective of both B 12 and folate status was elevated early in life, reaching a nadir at age 2 and then increasing towards adulthood., Conclusions: Continuous central 95 th percentile RI for holotranscobalamin, homocysteine and red cell folate for children ages 30 days to <18 years were established. Each marker shows dynamic changes throughout childhood and adolescence which will assist clinicians in more appropriately assessing B 12 and folate status in this population., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objectives: To investigate changes in von Willebrand factor (VWF) concentration, function, and multimers during pediatric extracorporeal membrane oxygenation (ECMO) and determine whether routine monitoring of VWF during ECMO would be useful in predicting bleeding., Design: Prospective observational study of pediatric ECMO patients from April 2017 to May 2019., Setting: The PICU in a large, tertiary referral pediatric ECMO center., Patients: Twenty-five neonates and children (< 18 yr) supported by venoarterial ECMO., Interventions: None., Measurements and Main Results: Arterial blood samples were collected within 24 hours pre-ECMO, daily for the first 5 days of ECMO, every second day until decannulation, and 24 hours post-ECMO. The STA R Max analyzer was used to measure VWF antigen (VWF:Ag) and ristocetin cofactor (VWF:RCo) activity. VWF collagen binding (VWF:CB) was measured using an enzyme-linked immunosorbent assay. VWF multimers were measured using the semi-automated Hydragel 11 VWF Multimer assay. Corresponding clinical data for each patient was also recorded. A total of 25 venoarterial ECMO patients were recruited (median age, 73 d; interquartile range [IQR], 3 d to 1 yr). The median ECMO duration was 4 days (IQR, 3-8 d) and 15 patients had at least one major bleed during ECMO. The percentage of high molecular weight multimers (HMWM) decreased and intermediate molecular weight multimers increased while patients were on ECMO, irrespective of a bleeding status. VWF:Ag increased and the VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios decreased while patients were on ECMO compared with the baseline pre-ECMO samples and healthy children., Conclusions: Neonates and children on ECMO exhibited a loss of HMWM and lower VWF:CB/VWF:Ag and VWF:RCo/VWF:Ag ratios compared with healthy children, irrespective of major bleeding occurring. Therefore, monitoring VWF during ECMO would not be useful in predicting bleeding in these patients and changes to other hemostatic factors should be investigated to further understand bleeding during ECMO., Competing Interests: Ms. Van Den Helm’s, Ms. Letunica’s, Mr. McCafferty’s, and Drs. Ignjatovic’s and Monagle’s institutions received funding from the Australian National Health and Medical Research Council. Dr. MacLaren disclosed that he serves on the Board of Directors of the Extracorporeal Life Support Organization. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2023 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)

Competing Interests: None declared.

Objectives: In adults, the elevation of cardiac troponin (cTn) above the 99th percentile upper reference limit defines myocardial injury. The use and interpretation of cTn in a paediatric population, however, is difficult given the 99th percentile for different assays is not well established. Using paediatric blood samples from healthy neonates, infants and children we derived continuous and partitioned 97.5th and 99th percentiles for the Ortho VITROS hs-TnI assay., Methods: A total of 328 samples for infants, children and adolescents aged 0-17.8 years were obtained. Age partitioned reference limits were derived in accordance with CLSI EP28-A3C. Continuous reference limits were established as described previously by the HAPPI Kids Study team., Results: hs-TnI as measured by the Ortho VITROS Assay is highly elevated above the adult 99th percentile at birth and declines to lower levels within the first 6 months of life. The 99th centile upper reference limit for ages 0-3 months was 72 ng/L (90% CI: 52-91) and 9 ng/L (90% CI: 5.2-17.4) for ages 3 months to 18 years. Continuous upper 99th centile reference limits were comparable., Conclusions: Partitioned and continuous 99th percentiles for hs-TnI were derived for the new Ortho VITROS assay in healthy neonates and older children. This will assist clinicians to appropriately assess for the presence of myocardial injury in this population., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Proteomics, the simultaneous study of all proteins in a given cell, tissue or organism, is an innovative approach used to identify novel markers for diagnosis, prognosis and the pathophysiological mechanisms associated with diseases. Proteomic methodologies have been used in a variety of contexts such as investigating changes in protein abundance that may occur with disease presence, the response to therapeutic treatments as well as the impacts of age on the plasma proteome.Over the last decade, significant technological advancements in proteomic techniques have resulted in an increase in the use of proteomics in thrombosis and hemostasis research, particularly in order to identify relevant and novel clinical markers associated with bleeding and thrombosis. This mini-review explores the use of proteomics in the setting of thrombosis and hemostasis from 2010-2020, across five main domains (platelets, blood clot composition, stroke, venous thromboembolism, and therapeutics), as well as provides insights into key considerations for conducting proteomic studies., Competing Interests: None declared., (Thieme. All rights reserved.)

COVID-19 has infected more than 275 million worldwide (at the beginning of 2022). Children appear less susceptible to COVID-19 and present with milder symptoms. Cases of children with COVID-19 developing clinical features of Kawasaki-disease have been described. Here we utilise Mass Spectrometry proteomics to determine the plasma proteins expressed in healthy children pre-pandemic, children with multisystem inflammatory syndrome (MIS-C) and children with COVID-19 induced ARDS. Pathway analyses were performed to determine the affected pathways. 76 proteins are differentially expressed across the groups, with 85 and 52 proteins specific to MIS-C and COVID-19 ARDS, respectively. Complement and coagulation activation are implicated in these clinical phenotypes, however there was significant contribution of FcGR and BCR activation in MIS-C and scavenging of haem and retinoid metabolism in COVID-19 ARDS. We show global proteomic differences in MIS-C and COVID-ARDS, although both show complement and coagulation dysregulation. The results contribute to our understanding of MIS-C and COVID-19 ARDS in children., (© 2022. The Author(s).)

Coronavirus disease 2019 (COVID-19) patients have increased thrombosis risk. With increasing age, there is an increase in COVID-19 severity. Additionally, adults with a history of vasculopathy have the highest thrombotic risk in COVID-19. The mechanisms of these clinical differences in risk remain unclear. Human umbilical vein endothelial cells (HUVECs) were infected with SARS-CoV-2, influenza A/Singapore/6/86 (H1N1) or mock-infected prior to incubation with plasma from healthy children, healthy adults or vasculopathic adults. Fibrin on surface of cells was observed using scanning electron microscopy, and fibrin characteristics were quantified. This experiment was repeated in the presence of bivalirudin, defibrotide, low-molecular-weight-heparin (LMWH) and unfractionated heparin (UFH). Fibrin formed on SARS-CoV-2 infected HUVECs was densely packed and contained more fibrin compared to mock-infected cells. Fibrin generated from child plasma was the thicker than fibrin generated in vasculopathic adult plasma ( p  = 0.0165). Clot formation was inhibited by LMWH (0.5 U/ml) and UFH (0.1-0.7 U/ml). We show that in the context of the SARS-CoV-2 infection on an endothelial culture, plasma from vasculopathic adults produces fibrin clots with thinner fibrin, indicating that the plasma coagulation system may play a role in determining the thrombotic outcome of SARS-CoV-2 infection. Heparinoid anticoagulants were most effective at preventing clot formation., Competing Interests: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (© 2022 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Objectives: Children with very short telomeres commonly develop bone marrow failure and other severe diseases. Identifying the individuals with short telomeres can improve outcome of bone marrow transplantation, with accurate diagnosis requiring the use of age-matched reference intervals (RIs). This study aimed to establish RIs for telomere length (TL) in children using three commonly used methods for TL measurement., Methods: Healthy children aged 30 days to 18 years were recruited for assessment using age as a continuous variable. Venous blood samples were collected and leukocyte TL was measured using terminal restriction fragment (TRF) analysis, quantitative PCR (QPCR) and flow cytometry with fluorescence in situ hybridization (Flow-FISH). Fractional polynomial model and quantile regression were performed to generate continuous RIs. Factors that might contribute to variation in TL, such as gender, were also examined., Results: A total of 212 samples were analyzed. Continuous RIs are presented as functions of age. TRF analysis and QPCR showed significant negative correlation between TL and age (r=-0.28 and r=-0.38, p<0.001). In contrast, Flow-FISH showed no change in TL with age (r=-0.08, p=0.23). Gender did not have significant influence on TL in children., Conclusions: This study provides three options to assess TL in children by establishing method-specific continuous RIs. Choosing which method to use will depend on several factors such as amount and type of sample available and required sensitivity to age-related change., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Introduction: To facilitate best possible patient care, reference intervals (RIs) adopted by a laboratory must be appropriate for the population demographics and, where applicable, the analytical principle and/or the analytical instrument used. While guidelines from the Clinical and Laboratory Standard Institute (CLSI) recommend a validation process for discrete RIs, there are no current recommendations for the validation process for continuous RIs. This study aimed to validate recently published, HAPPI Kids continuous RIs, in a routine laboratory., Methods: Initially, the difference in test results between the primary study laboratory that contributed to previous RIs development and a routine laboratory was assessed using specimens from 77 children tested in both laboratories using the Siemens ADVIA 1800 or Centaur/XP/XPT. Later, validation of the HAPPI Kids RIs was undertaken using 279 pediatric samples tested on the same analyzer type in the routine laboratory. The previously published RIs were validated if more than 90% of results in the routine laboratory were within the RIs., Results: There was minimal evidence of clinically significant differences in test results between the primary and routine laboratories. The continuous RIs were validated after initial analysis for 16 of the 18 biochemistry analytes tested, and after secondary analysis for the remaining 2 analytes., Conclusion: This study validates the HAPPI Kids RIs in a routine laboratory, satisfying the laboratory accreditation requirements for evaluation, implementation, and sourcing of RIs. In addition, this study presents a modification of the current CLSI method for validation of continuous RIs that will benefit routine laboratories in general., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Serum cystatin C (CysC) is a promising biomarker of kidney function, which has higher accuracy and sensitivity when compared with creatinine. To better utilize serum CysC in clinical practice, this study aimed to establish continuous paediatric reference intervals (RIs) for serum CysC., Methods: The study subjects consisted of healthy term neonates and children aged 30 days to 18 years. Venous blood samples were collected and serum CysC levels were measured using the immunoturbidimetric measurement principle. Fractional polynomial regression model and quantile regression was applied in the statistical analysis to generate continuous RIs., Results: A total of 378 samples with equal numbers of males and females were analysed for serum CysC. No outliers were found in this analysis. The continuous RIs are presented as equations and graphical scatterplots., Conclusions: This study established continuous paediatric reference intervals (RIs) for serum CysC in healthy term neonates and children. The continuous RIs generated from this study show age-based dynamic changes as well as blood group and gender-specific differences for serum CysC. Graphical abstract.

Background: Age-specific reference intervals (RIs) have been developed for biochemistry analytes in children. However, the ability to interpret results from multiple laboratories for 1 individual is limited. This study reports a head-to-head comparison of reference values and age-specific RIs for 30 biochemistry analytes for children across 5 analyzer types., Methods: Blood was collected from healthy newborns and children 30 days to <18 years of age. Serum aliquots from the same individual were analyzed on 5 analyzer types. Differences in the mean reference values of the analytes by the analyzer types were investigated using mixed-effect regression analysis and by comparing maximum variation between analyzers with analyte-specific allowable total error reported in the Westgard QC database. Quantile regression was used to estimate age-specific RIs using power variables in age selected by fractional polynomial regression for the mean, with modification by sex when appropriate., Results: The variations of age-specific mean reference values between analyzer types were within allowable total error (Westgard QC) for most analytes, and common age-specific reference limits were reported as functions of age and/or sex. Analyzer-specific reference limits for all analytes on 5 analyzer types are also reported as functions of age and/or sex., Conclusions: This study provides quantitative and qualitative measures of the extent to which results for individual children can or cannot be compared across analyzer types, and the feasibility of RI harmonization. The reported equations enable incorporation of age-specific RIs into laboratory information systems for improving evidence-based clinical decisions in children., (© 2019 American Association for Clinical Chemistry.)

Introduction: The clinical interpretation of laboratory tests is reliant on reference intervals. However, the accuracy of a reference interval is dependent on the selected reference population, and in paediatrics, the ability of the reference interval to reflect changes associated with growth and age, as well as sex and ethnicity. Differences in reagent formulations, methodologies and analysers can also impact on a reference interval. To date, no direct comparison of reference intervals for common analytes using different analysers in children has been published. The Harmonising Age Pathology Parameters in Kids (HAPPI Kids) study aims to establish age-appropriate reference intervals for commonly used analytes in the routine clinical care of neonates and children, and to determine the feasibility of paediatric reference interval harmonisation by comparing age-appropriate reference intervals in different analysers for multiple analytes., Methods and Analysis: The HAPPI Kids study is a prospective cross-sectional study, collecting paediatric blood samples for analysis of commonly requested biochemical, immunological and haematological tests. Venous blood samples are collected from healthy premature neonates (32-36 weeks of gestation), term neonates (from birth to a maximum of 72 hours postbirth) and children aged 30 days to ≤18 years (undergoing minor day surgical procedures). Blood samples are processed according to standard laboratory procedures and, if not processed immediately, stored at -80°C. A minimum of 20 samples is analysed for every analyte for neonates and then each year of age until 18 years. Analytical testing is performed according to the standard operating procedures used for clinical samples. Where possible, sample aliquots from the same patients are analysed for an analyte across multiple commercially available analysers., Ethics and Dissemination: The study protocol was approved by The Royal Children's Hospital, Melbourne, Ethics in Human Research Committee (34183 A). The study findings will be published in peer-reviewed journals and shared with clinicians, laboratory scientists and laboratories., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Human blood plasma is a complex biological fluid containing soluble proteins, sugars, hormones, electrolytes, and dissolved gasses. As plasma interacts with a wide array of bodily systems, changes in protein expression, or the presence or absence of specific proteins are regularly used in the clinic as a molecular biomarker tool. A large body of literature exists detailing proteomic changes in pathologic contexts, however little research has been conducted on the quantitation of the plasma proteome in age-specific, healthy subjects, especially in pediatrics. In this study, we utilized SWATH-MS to identify and quantify proteins in the blood plasma of healthy neonates, infants under 1 year of age, children between 1-5 years, and adults. We identified more than 100 proteins that showed significant differential expression levels across these age groups, and we analyzed variation in protein expression across the age spectrum. The plasma proteomic profiles of neonates were strikingly dissimilar to the older children and adults. By extracting the SWATH data against a large human spectral library we increased protein identification more than 6-fold (940 proteins) and confirmed the concentrations of several of these using ELISA. The results of this study map the variation in expression of proteins and pathways often implicated in disease, and so have significant clinical implication., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Post-translational modifications (PTMs) such as glycosylation and phosphorylation play an important role on the function of haemostatic proteins and are critical in the setting of disease. Such secondary level changes to haemostatic proteins have wide ranging effects on their ability to interact with other proteins. This review aimed to summarize the knowledge of the common PTMs associated with haemostatic proteins and the implications of such modifications on protein function. Haemostatic proteins that represent the main focus for studies specific to PTMs are von Willebrand factor, tissue factor, factor VIII, antithrombin and fibrinogen. These proteins are susceptible to PTMs by glycosylation, phosphorylation, sulphation, citrullination and nitration, respectively, with a significant impact on their function. During synthesis, vWF must undergo extensive PTMs, with N-linked glycosylation being the most common. Increased phosphorylation of tissue factor results in increased affinity for platelets to the vessel endothelium. Citrullination of antithrombin leads to an increased anticoagulant function of this protein and therefore an anticoagulant state that inhibits clot formation. On the contrary, nitration of fibrinogen has been shown to result in a prothrombotic state, whilst sulphation is required for the normal function of Factor VIII. From this review, it is evident that PTMs of haemostatic proteins as a change in protein structure at a secondary level greatly influences the behaviour of the protein at a tertiary level.

Introduction: Infants and children have a lower incidence of thrombosis compared with adults. Yet, the mechanism of blood clot formation and structure in infants and children, as the end product of coagulation, has not been studied. This study aimed to establish differences in the mechanism of thrombin generation, fibrin clot formation and response to thrombolysis in infants and children compared with adults., Materials and Methods: We studied thrombin generation, fibrin clot formation, structure and fibrinolysis in healthy infants, children and adults., Results: Younger populations had a decreased potential to generate thrombin, at a slower velocity compared with adults, correlating positively with age. Clot formation at venous shear rate was decreased in infants and children compared with adults, with increased time for fibrin formation, decreased fibrin formation velocity, resulting in decreased tendency for fibrin formation in younger populations. These differences were less pronounced at arterial shear rate. Studies of the fibrin clot structure in paediatric age groups showed a significantly larger pore size compared with adults, suggestive of a clot that is less resistant to fibrinolysis. The presence of tissue plasminogen activator (tPA) resulted in a significant decrease in the pore size of infants and children, but not in adults., Conclusions: This is the first study to suggest that the mechanism of blood clot formation and nanostructure, as well as response to thrombolytic therapy is different in infants and children compared with adults., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Major age-related diseases such as cardiovascular disease and cancer are the primary causes of morbidity and mortality in Australia and worldwide. In our recent study characterising differences in the plasma proteome between healthy children and adults, a large number of proteins differentially expressed with age were found to be of platelet origin. This study aimed to characterise differences in the resting platelet proteome and the platelet releasate of healthy children compared to healthy adults. This study represents the setup of a procedure for the proteomic analysis of platelets from children. Differentially expressed platelet proteins were identified using Two-dimensional Differential In-Gel Electrophoresis and mass spectrometry. Significant differences in the expression of nine proteins (1.1%) in the resting platelet proteome were observed in children compared to adults. Serotransferrin, fibrinogen alpha chain, glyceraldehyde-3 phosphate dehydrogenase, serum albumin, transgelin-2, calponin-2/LIM and SH3 domain protein 1 and human chorionic gonadotropin 2039797 were up-regulated, whereas thrombospondin-1 was down-regulated in children. Eleven proteins (1.5%) were differentially expressed in the platelet releasate of children compared to adults, where transferrin was also upregulated and TSP-1 was down regulated. Identified proteins are involved in processes including tissue and organ development, cell proliferation regulation and angiogenesis. Our results provide novel insights into platelet physiology as well as growth, development and ageing in healthy individuals., Biological Significance: The incidence of major diseases such as cardiovascular disease (CVD) and cancer increase with increasing age and are the major causes of morbidity and mortality both in Australia and worldwide. As the aged population continues to increase dramatically, so too will the financial strains associated with the long term care of the elderly population. Compared to adults, children have a significantly lower incidence of major diseases such as thromboembolic disease. This suggests that children have a protective mechanism against the development of disease. Therefore, studies focussing on the molecular changes of proteins, the machinery of the cell, between children and adults are the key to determining the underlying mechanisms responsible for the onset of major diseases. A well-defined example of how protein expression can change with age is that of the plasma proteome. Significant differences in the expression of numerous plasma proteins between healthy children and adults have been recently demonstrated. Interestingly, a large number of differentially expressed proteins were found to be of platelet origin. This finding forms the basis for the current study, presenting as strong evidence for the age-specific differences of the platelet proteome., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Remote ischemic preconditioning (RIPC) has been applied in paediatric cardiac surgery. We have demonstrated that RIPC induces a proteomic response in plasma of healthy volunteers. We tested the hypothesis that RIPC modifies the proteomic response in children undergoing Tetralogy of Fallot (TOF) repair., Methods and Results: Children (n=40) were randomized to RIPC and control groups. Blood was sampled at baseline, after cardiopulmonary bypass (CPB) and 6, 12 and 24h post-CPB. Plasma was analysed by liquid chromatography mass spectrometry (LC-MS) in an untargeted approach. Peptides demonstrating differential expression (p<0.01) were subjected to tandem LC-MS/MS and protein identification. Corresponding proteins were identified using the NCBI protein database. There was no difference in age (7.3±3.5vs6.8±3.6 months)(p=0.89), weight (7.7±1.8vs7.5±1.9 kg)(p=0.71), CPB time (104±7vs94±7 min)(p=0.98) or aortic cross-clamp time (83±22vs75±20 min)(p=0.36). No peptides were differentially expressed at baseline or immediately after CPB. There were 48 peptides with higher expression in the RIPC group 6h post-CPB. This was no longer evident at 12 or 24h, with one peptide down-regulated in the RIPC group. The proteins identified were: inter-alpha globulin inhibitor (42.0±11.8 vs 820.8±181.1, p=0.006), fibrinogen preproprotein (59.3±11.2 vs 1192.6±278.3, p=0.007), complement-C3 precursor (391.2±160.9 vs 5385.1±689.4, p=0.0005), complement C4B (151.5±17.8 vs 4587.8±799.2, p=0.003), apolipoprotein B100 (53.4±8.3 vs 1364.5±278.2, p=0.005) and urinary proteinase inhibitor (358.6±74.9 vs 5758.1±1343.1, p=0.009). These proteins are involved in metabolism, haemostasis, immunity and inflammation., Conclusions: We provided the first comprehensive analysis of RIPC-induced proteomic changes in children undergoing surgery. The proteomic changes peak 6h post-CPB and return to baseline within 24h of surgery., Trial Registration: ACTR.org.au ACTRN12610000496011.

Protein C (PC) is a 62-kDa vitamin K-dependent plasma zymogen which, after activation to serine protease, plays an important role in the physiologic regulation of blood coagulation. Given that PC is one of the major naturally occurring inhibitors of coagulation, acquired or hereditary deficiencies of this protein result in excessive thrombin generation. As a vast array of mutations are responsible for hereditary PC deficiencies, screening for their presence by DNA testing would require sequencing each entire gene involving numerous exons. Moreover, the knowledge of the gene mutation does not offer any benefit in the treatment of thrombophilic families, so the routine molecular characterization is not indicative. These defects are detected by functional or immunological assays. Measurement of PC activity is essential to identify subjects with both type I and type II PC defects. There is no need to routinely perform PC immunological assays. However, they are useful in order to distinguish type I from type II PC hereditary deficiency.

Protein S (PS) is a vitamin K-dependent plasma glycoprotein. Around 60-70% of PS in plasma is noncovalently bound to C4-binding protein (C4BP). Free PS functions as a cofactor that enhances the activity of activated protein C (APC) in the proteolytic degradation of activated factors V and VIII. PS also has a more recently described APC-independent ability to directly inhibit prothrombinase and tenase by direct binding of activated factors V, VIII, and X. Given that PS is one of the major naturally occurring inhibitors of coagulation, acquired or hereditary deficiencies of this protein result in excessive thrombin generation. As a vast array of mutations are responsible for hereditary PS deficiencies, screening for their presence by DNA testing would require sequencing each entire gene involving numerous exons. Moreover, the knowledge of the gene mutation does not offer any benefit in the treatment of thrombophilic families, so the routine molecular characterization is not indicative. These defects are detected by functional or immunological assays for free and total PS forms. Given that functional PS assays may detect some forms of PS deficiency that free PS immunoassays may miss, it is recommended to include them for initial testing along with immunoassays for free PS, although they should be used with caution. Functional PS assays are subject to multiple interference. For example in the presence of lupus anticoagulant (LA), only free PS immunoassays are recommended for initial testing. PS antigen assays are more popular with most laboratories.

Antithrombin (AT) is a heparin cofactor and a member of the serine protease inhibitor family (serpin). The mature AT molecule is composed of 432 amino acids and it is produced mainly in the liver. Initially, several different AT activities in plasma were reported, leading to the classification of antithrombin in a range from I to IV. It was subsequently shown that these various antithrombin activities were the function of one molecule, antithrombin III, whose name was reduced to antithrombin at the meeting of the International Society in Thrombosis and Haemostasis in 1993. AT is an important protease inhibitor of thrombin and factor Xa. However, AT is also able to inhibit factors IXa, XIa, XIIab, kallikrein, and plasmin. Given that AT is one of the major naturally occurring inhibitors of coagulation, acquired or hereditary deficiencies of this protein result in excessive thrombin generation. As a vast array of mutations are responsible for hereditary AT deficiencies, screening for their presence by DNA testing would require sequencing each entire gene involving numerous exons. Moreover, the knowledge of the gene mutation does not offer any benefit in the treatment of affected families, so the routine molecular characterization is not indicative. These defects are detected by functional or immunological assays. AT amidolytic assays are recommended for initial testing for AT deficiency. There is no need to routinely perform AT immunological assays. However, they are useful in order to distinguish type I from type II hereditary AT deficiency.