ABSTRACT
Background and Aims
Children's coagulation system undergoes dynamic changes during development, necessitating age‐specific reference intervals (RIs). This study aimed to establish RIs for 16 coagulation parameters in Chinese children aged 0–18 years using the Sysmex CN‐6000 analyzer.
Methods
We conducted a cross‐sectional study in children aged 0 day to 18 years who met the inclusion and exclusion criteria. Participants were stratified into six age groups (0–30 days, 1–6 months, 6–12 months, 1–3 years, 3–12 years, and 12–18 years) and blood samples were collected. Routine parameters (APTT, PT, PT‐INR, TT, Fibrinogen [Fib]), thrombotic markers (AT‐III, D‐Dimer [D‐Di], Fibrin Degradation Products [FDP]), and coagulation factors (II, V, VII, VIII, IX, X, XI, XII) were analyzed using the Sysmex CN‐6000 analyzer. RIs and their 90% confidence intervals (CIs) were calculated using the non‐parametric method (for n ≥ 120) or the robust method (for n < 120).
Results
Neonates exhibited distinct coagulation profiles, including prolonged APTT (22.7 s–46.5 s) and TT (16.2 s–20.8 s), reduced levels of anticoagulant and coagulation factors (e.g., AT‐III: 23.1%–91.3%, factor II: 26.5%–77.5%), and elevated fibrinolytic markers (D‐Di ≤ 1.41 μg/mL; FDP ≤ 4.44 μg/mL). Age‐dependent trends were evident: APTT shortened until adolescence (24.7 s–32.9 s), while factor IX increased progressively (16.3% – 43.2% in neonates vs. 43.6%–108.6% in adolescents). Novel RIs for neonatal D‐Di and FDP were established, addressing a critical gap in developmental hemostasis. Sex‐specific differences in TT were observed but deemed clinically insignificant.
Conclusion
Age‐specific RIs are essential for children. This study provides the first comprehensive RIs for Sysmex CN‐6000 in Chinese children, aiding in the diagnosis and management of coagulation disorders in pediatrics.
Keywords: age‐specific, coagulation parameters, neonates, pediatrics, reference intervals
1. Introduction
Coagulation function tests are the most commonly used diagnostic tests in clinical practice, playing a crucial role in the assessment of coagulation function, screening for bleeding or thrombotic disorders, and monitoring of anticoagulant or thrombolytic therapies. However, when it comes to the interpretation of these tests in the pediatric population, it remains challenging due to the ongoing maturation of the coagulation system in children, especially neonates and infants, which presents distinct hemostatic profiles compared to adults [1].
Since the 1980s when Andrew, M et al. proposed the concept of developmental hemostasis, the idea that coagulation function is a dynamically evolving progress has been widely accepted [2, 3]. Clinical work to establish reference intervals (RIs) for children of different age groups have been carried out in several countries, such as France, the United States, Austria, and Italy [4, 5, 6]. In China, clinical research on coagulation parameters RIs for children at different age stages are relatively scarce, and mainly focused on the routine coagulation indicators such as the Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT), Fibrinogen (Fib), and Thrombin Time (TT) [7, 8]. Study on the RIs for coagulation factors and fibrinolytic markers are still limited. Besides, routine coagulation parameters (APTT, PT, Fib, and TT) reflect the overall state of the blood coagulation system, which involves multiple coagulation factors. From the perspective of developmental hemostasis, the levels of most coagulation proteins, especially the vitamin K‐dependent factors, in children also dynamically change. Therefore, appropriate age‐specific RIs for coagulation factors are also needed in case of misdiagnosis.
Currently, most laboratories in China adopt RIs recommended by manufacturers or the Chinese National Guide to Clinical Laboratory Operational Procedures with fewer institutions use self‐established RIs based on their laboratory conditions [9]. Moreover, most of these RIs are for adult populations. Considering the special physiological characteristics of hemostasis in children, it is necessary for laboratories to establish suitable coagulation parameters RIs for children at different age stages to enhance clinical accuracy in diagnosing and managing pediatric coagulation disorders.
2. Materials and Methods
2.1. Study Design
This cross‐sectional study was conducted on Shenzhen Children's Hospital, China, from January 2024 to November 2024. The study was approved by the Medical Ethics Committee of Shenzhen Children's Hospital (Approval No. 202501302) and adhered fully to the principles of the Declaration of Helsinki. Written informed consent was obtained from all subjects or patients' guardians involved in the study. The reporting followed the STROBE guideline to ensure the quality of the observational research.
2.2. Study Population
Apparently healthy children aged from 0 days to 18 years were included in our study. The inclusion criteria of the study were children with normal complete blood count (CBC) and normal liver and kidney function, confirmed by laboratory tests including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (Crea). Patients with allergic/pulmonary/rheumatic/endocrine diseases, hypertension, diabetes, heart diseases, malignant tumors, familial hereditary diseases related to bleeding or thrombosis, infections or surgical/traumatic history within the last 30 days, oral anticoagulant medications or blood transfusion history within the last 6 months were excluded. The newborns were healthy full‐term neonates without any bleeding or thrombotic disorders, anticoagulant treatment, intrapartum asphyxia or a history of familial bleeding disorders, and were not administered with vitamin K at birth routinely. We sought to enroll a study population with a sex ratio as close to 1:1 as possible.
Children were stratified into six age groups as follows: (i) 0 day–30 days (denoted as 0–30 d), (ii) 1 month –≤ 6 months (denoted as 1–6 m), (iii) 6 months –≤ 12 months (denoted as 6–12 m), (iv) 1 year –≤ 3 years (denoted as 1–3 y), (v) 3 years –≤ 12 years (denoted as 3–12 y), and (vi) 12 years –≤ 18 years (denoted as 12–18 y).
2.3. Sample Collection
Specimen collection, delivery, and storage were strictly carried out under the guideline of Collection and processing of blood specimens for testing plasma‐based coagulation assays (WS/T 359‐2011) which were based on the CLSI guideline H21 [10]. A total of 1.8 mL of venous blood was collected and placed into 0.2 mL of 3.2% (109 mmol/L) sodium citrate, mixed thoroughly with blood at a 1:9 ratio, and sent for testing immediately. The specimens were centrifuged at 2000g for 15 min. Any specimens with clots, hemolysis, lipemia, or other abnormalities were excluded. Plasma for further analyzing RIs for other coagulation parameters were separated and stored at −80°C within 4 h after blood collection. Before testing, the plasma was thawed at 37°C for 5 min, mixed well, and tested promptly on the analyzer.
2.4. Coagulation Parameters Testing
Samples were analyzed on the Sysmex CN‐6000 automatic coagulation analyzer (Japan) along with its corresponding reagents. Internal quality control and external quality assessment were strictly carried out to ensure the accuracy of the test results. Routine coagulation parameters (APTT, PT, TT, Fib) and coagulation factors (II, V, VII, VIII, IX, X, XI, XII) were measured using the clotting methods. D‐Dimer (D‐Di) and Fibrin Degradation Products (FDP) were measured using the immunoturbidimetric methods. Antithrombin III (AT‐III) was measured using the chromogenic substrate method. All tests were strictly performed according to the operating steps described in the reagent instructions.
The reagents used in our study included Thromborel S (PT), Dade Actin FSL Activated PTT Reagent (APTT), Test Thrombin Reagent (TT), Dade Thrombin Reagent (Fib), Berichrom Antithrombin III (AT‐III), INNOVANCE D‐Dimer (D‐Di), LIASAUTO‐FDP (FDP), Coagulation factor deficient Plasmas and Dade Actin FSL Activated PTT Reagent (coagulation factors VIII, IX, XI and XII), Coagulation factor deficient Plasmas and Thromborel S (coagulation factors II, V, VII and X) from Siemens Healthcare Diagnostics, Marburg, Germany.
2.5. Statistical Analysis
2.5.1. Treatment of Outliers and Assessment of RIs Partitioning
The sample sizes were determined according to CLSI EP28‐A3c [11]. Statistical analyses were performed using SPSS Version 20.0 (IBM, New York, USA) and GraphPad Prism version 8 (GraphPad Software Inc, Boston, USA) was used to draw the plot. The Shapiro–Wilk test was used to assess the normality of the data. Skewed data were transformed into an approximately normal distribution using the Box‐Cox method via Stata Software Version 18.0 (StataCorp, College Station, TX, USA) for further outlier removal. According to the CLSI EP28‐A3c [11], outliers were removed using the Tukey method. The necessity of establishing separate RIs by sex or age was assessed using the Mann–Whitney U test (for sex group comparison) and the Kruskal–Wallis tests (for age group comparison). The statistical tests were two‐sided, with the significance level set at p < 0.05. Following a significant Kruskal–Wallis tests, post hoc pairwise comparisons were conducted. To account for multiple comparisons, a Bonferroni correction was applied to the post hoc tests. The significance level (α) was set at 0.05, and with five independent tests (comparisons between adjacent age groups) performed, the corrected significance threshold was p < 0.01. If the differences in the group comparisons were found statistically significant, it is assumed that separate RIs for the subclasses groups are warranted.
2.5.2. Calculation of RIs and Reference Limit's CIs
As recommended by CLSI EP28‐A3c [11], lower and upper reference limits (RIs) for coagulation parameters (excluding D‐Di and FDP) were defined as values at the 2.5th and 97.5th percentiles (P2.5 − P97.5), respectively. The upper limits (RIs) for D‐Di and FDP were defined as the values at 95th percentiles (≤ P95) since D‐Di and FDP only have clinical significance in positive values.
Coagulation RIs were calculated using MedCalc Statistical Software Version 23.1.1 (MedCalc Software, Ostend, Belgium). Based on the sample sizes, we applied the nonparametric method (for n ≥ 120) or the robust method (for n < 120). The corresponding 90% confidence intervals (CIs) for reference limits were also provided by ranked observation method (the non‐parametric method, for n ≥ 120) or bootstrap percentile method using 1000 bootstrap replicates (the robust method of Horn and Pesce, for n < 120) [12, 13]. Details of participants selection and the data analysis process are illustrated in Figure 1.
Figure 1.
Flowchart of participant selection and data analysis.
3. Results
3.1. Characteristics of the Study Participants
The study population, recruited from January 2024 to November 2024, were mainly from the Urology Department and Day Surgery Center who were undergoing preoperative screening for elective surgery, as well as healthy full‐term newborns from the Neonatology Department. A total of 1220 healthy individuals (618 males and 602 females) were included for the retrospective analysis of APTT, PT, PT‐INR, TT and Fib. Besides, 438 cases (230 males and 208 females) were included for the analysis of AT‐III, D‐Di, FDP, and the eight coagulation factors (II, V, VII, VIII, IX, X, XI, XII).
In our clinical practice, the routine coagulation tests (APTT, PT, TT, Fib), fibrinolytic marker tests (D‐Di and FDP) and anticoagulant protein test (AT‐III) are performed on fresh samples. While coagulation factor tests are performed on frozen samples. Children scheduled for minor surgery in our hospital were routinely monitored for the four coagulation tests (APTT, PT, TT and Fib). Retrospective analysis of RIs for AT‐III, D‐Di and FDP are not applicable. Based on researches that AT‐III, D‐Di and FDP maintain stability when frozen at ≤− 70°C for up to 24 months [10], we established the RIs for AT‐III, D‐Di and FDP from frozen samples.
3.2. Distributions and RIs for Coagulation Parameters
Distributions of the 16 coagulation parameters were provided in Figure 2, while their median and interquartile range (Q1, Q3) were detailed in Supporting Information S1: Tables S1 and S2. According to the CLSI EP28‐A3c guideline, we calculated the RIs for the 16 coagulation parameters and detailed them in Tables 1 and 2.
Figure 2.
Distribution of the 16 coagulation parameters in different age partitions. (A) Routine coagulation tests: activated partial thromboplastin time (APTT), prothrombin time (PT), prothrombin time ‐ International Normalized Ratio (PT‐INR), thrombin time (TT) and fibrinogen (Fib). (B) Anticoagulant factors: antithrombin III (AT‐III). (C) Vitamin K‐dependent coagulation factors: factor II, VII, IX and X. (D) Fibrinolytic markers: D‐Dimer (D‐Di) and Fibrin Degradation Products (FDP). (E) Vitamin K‐independent coagulation factors: factor V, VIII, XI and XII.
Table 1.
Age‐specific RIs (P2.5 ~ P97.5) and 90% CIs for reference limits for coagulation parameters in children aged 0–18 years.
| Analyte (Unit) | Partition (M/F) | P2.5 | P97.5 | 90% CI for P2.5 | 90% CI for P97.5 |
|---|---|---|---|---|---|
| APTT (s) | 0–30days (15/20) | 22.7 | 46.5 | 20.4–25.2 | 43.0–49.2 |
| 1–6 months (79/72) | 24.1 | 38.1 | 22.9 ~ 24.9 | 35.9–38.4 | |
| 6 months‐3 years (255/245) | 23.4 | 32.6 | 23.0–23.8 | 31.9–33.2 | |
| 3–18 years (269/265) | 24.7 | 32.9 | 24.1–24.9 | 32.3–33.3 | |
| PT (s) | 0 day–6 months (94/92) | 10.2 | 13.0 | 9.9–10.3 | 12.6–13.3 |
| 6 months–3 years (255/245) | 10.1 | 12.4 | 9.8–10.1 | 12.2–12.6 | |
| 3–12 years (133/131) | 10.3 | 12.4 | 10.1–10.4 | 12.3–12.7 | |
| 12–18 years (136/134) | 10.4 | 13.0 | 10.3–10.6 | 12.8–13.3 | |
| PT‐INR | 0 day–6 months (94/92) | 0.87 | 1.12 | 0.85–0.88 | 1.09–1.15 |
| 6 months–3 years (255/245) | 0.87 | 1.07 | 0.84–0.87 | 1.05–1.09 | |
| 3–12 years (133/131) | 0.88 | 1.07 | 0.87–0.89 | 1.06–1.10 | |
| 12–18 years (136/134) | 0.89 | 1.13 | 0.88–0.91 | 1.11–1.15 | |
| TT (s) | 0 day–12 months (219/209) | 16.2 | 20.8 | 15.9–16.4 | 20.4–21.1 |
| 1–18 years (399/393) | 15.8 | 18.4 | 15.7–15.9 | 18.2–18.5 | |
| Fib (g/L) | 0–30 days (15/20) | 1.48 | 3.24 | 1.26–1.70 | 3.02–3.42 |
| 1–12 months (204/189) | 1.29 | 3.03 | 1.21–1.33 | 2.96–3.19 | |
| 1–3 years (130/128) | 1.52 | 3.58 | 1.50–1.58 | 3.19–3.67 | |
| 3–18 years (269/265) | 1.74 | 3.65 | 1.65–1.80 | 3.57–3.74 | |
| AT‐III (%) | 0–30 days (12/9) | 23.1 | 91.3 | 13.3–35.6 | 79.3–101.0 |
| 1–6 months (34/18) | 61.3 | 117.0 | 55.6–68.1 | 112.2–122.3 | |
| 6 months–12 years (141/141) | 81.8 | 129.0 | 79.3–87.8 | 124.9–132.5 | |
| 12–18 years (43/40) | 71.7 | 116.7 | 65.2–77.5 | 114.1–119.0 | |
| II (%) | 0–30 days (12/9) | 26.5 | 77.5 | 19.8–34.7 | 68.7–85.2 |
| 1–6 months (34/18) | 46.5 | 113.6 | 40.7–53.1 | 106.0–119.8 | |
| 6 months–18 years (183/181) | 72.1 | 127.2 | 66.7–74.0 | 123.2–134.2 | |
| V (%) | 0 day–3 years (130/111) | 57.0 | 156.5 | 44.5–69.1 | 151.3–164.8 |
| 3–18 years (100/97) | 55.9 | 139.1 | 49.3–64.9 | 132.4–141.4 | |
| VII (%) | 0–18 years (229/207) | 54.6 | 113.8 | 48.7–57.5 | 108.8–119.0 |
| VIII (%) | 0 day–3 years (130/111) | 41.0 | 140.6 | 40.3–41.8 | 133.5–159.6 |
| 3–18 years (100/97) | 46.2 | 139.6 | 41.4–49.7 | 133.5–156.0 | |
| IX (%) | 0–30 days (12/9) | 16.3 | 43.2 | 12.9–20.7 | 38.8–47.3 |
| 1–6 months (34/18) | 19.4 | 64.3 | 15.1–24.2 | 59.0–69.0 | |
| 6–12 months (29/35) | 31.0 | 79.6 | 27.2–35.7 | 75.1–83.3 | |
| 1–3 years (55/49) | 44.4 | 81.5 | 42.6–46.3 | 77.6–85.3 | |
| 3–12 years (57/57) | 44.9 | 92.4 | 42.9–46.9 | 87.7–97.1 | |
| 12‐18 years (43/40) | 43.6 | 108.6 | 39.1–48.5 | 102.9–113.7 | |
| X (%) | 0‐30 days (12/9) | 27.4 | 103.3 | 17.7–38.2 | 90.3–114.5 |
| 1–6 months (34/17) | 53.5 | 111.0 | 48.3–60.2 | 105.2–116.0 | |
| 6 months–18 years (184/181) | 66.9 | 126.1 | 61.9–68.5 | 123.6–129.2 | |
| XI (%) | 0–30 days (12/9) | 15.7 | 76.3 | 7.4–26.1 | 66.2–86.0 |
| 1–6 months (34/18) | 41.7 | 117.4 | 38.0–46.6 | 104.9–130.1 | |
| 6–12 months (29/35) | 68.5 | 142.8 | 64.5–73.5 | 132.8–151.7 | |
| 1–12 years (112/106) | 61.5 | 148.4 | 59.1–78.5 | 142.7–165.8 | |
| 12–18 years (43/40) | 55.9 | 131.4 | 50.8–62.1 | 124.8–136.9 | |
| XII (%) | 0–30 days (12/9) | 11.2 | 54.7 | 9.0–14.5 | 42.3–67.3 |
| 1–6 months (34/18) | 19.3 | 95.9 | 16.7–22.2 | 74.4–114.3 | |
| 6 months–18 years (184/181) | 31.5 | 109.3 | 28.9–34.0 | 104.0–123.0 |
Abbreviations: F, female; M, male.
Table 2.
Age‐specific RIs (≤ P95) and 90% CIs for reference limits for coagulation parameters in children aged 0–18 years.
| Analyte (Unit) | Partition (M/F) | P95 | 90% CI for P95 |
|---|---|---|---|
| D‐Di (μg/mL) | 0–30 days (12/9) | 1.41 | 1.13–2.09 |
| 1 month–18 years (218/199) | 0.63 | 0.56–0.72 | |
| FDP (μg/mL) | 0–30 days (12/9) | 4.44 | 3.75–5.20 |
| 1 month–18 years (218/199) | 3.06 | 2.78–3.28 |
Abbreviations: F, female; M, male.
From the data, age dependency of coagulation parameters in pediatric population were observed in our study especially the APTT, which was significantly prolonged in the neonates (median: 33.7 s; RI: 22.7 s – 46.5 s) compared to adolescents (median: 28.0 s; RI: 24.7 s–32.9 s). Longer TT was demonstrated in the younger children within the first year of age with RI of 16.2 s–20.8 s, and then it shortened with RI of 15.8 s–18.4 s. Changes of PT were not obvious as APTT and TT. Fib exhibits a U‐shaped curve across the age groups, characterized by elevated levels in the neonatal period, followed by a decline during 1–6 months of age, and a subsequent increase until adolescence. Fibrinolytic markers (D‐Di and FDP) were found elevated in the neonates ((D‐Di ≤ 1.41 μg/mL; FDP ≤ 4.44 μg/mL). In the anticoagulation system, AT‐III exhibits the lowest levels in neonates (median: 60.7%, RI: 23.1%–91.3%), gradually rising after the first month of life and stabilizing by adolescence.
From the coagulation factor profiles, vitamin K‐dependent factors (II, VII, IX, and X) are at relatively low levels in neonates and gradually increase to adult levels by adolescence. Factor II and X share the same trend of change, as both increase gradually during the first year of life and then remain stable. Factor IX increased progressively from 30.5% (median) in neonates to 75.1% (median) in adolescents, with RI of (16.3–43.2)% in neonates and RI of (43.6–108.6)% in adolescents. Contact factors XI and XII also increase with age from median of 47.3% and 24.3% in neonates to median of 90.9% and 57.7% in adolescents, respectively. Factor V shows an overall trend of increasing first and then decreasing, while factor VIII did not change significantly with age.
Additionally, we found that the sex‐specific difference in TT remained statistically significant with the p‐value of 0.002 (Z = −3.025, effect size r = 0.087, Supporting Information S1: Table S3). According to Sinton et al. [14], separate RIs would not be estimated unless the difference between the subclass means is at least 25% as large as the 95% RI estimated from the combined (overall) sample of reference subjects. Thus, we did not set separate RIs for TT of different sexes. Due to the insufficient sample sizes, we did not compare the sex differences of AT‐III, D‐Di, FDP and the eight coagulation factors.
4. Discussion
The Sysmex CN‐6000 used in our laboratory is a new type of automated multi‐parameter coagulation analyzer that performs clotting, chromogenic and immunological assays, and platelet aggregation tests in a single system. Up to now, there are little literature reports on the coagulation‐related RIs for this instrument, especially among the pediatric population. We have observed that the RIs provided by the manufacturer were slightly different from what is seen in our clinical practice. Thus, it is necessary for our laboratory to establish RIs for coagulation parameters based on our circumstances. According to CLSI EP28‐A3c, establishing RIs by direct sampling techniques are recommended. However, it is a rather challenging endeavor in the aspect of establishing RIs for pediatric population. Here, we used indirect sampling techniques instead. We retrospectively analyzed the results of APTT, PT, PT‐INR, TT and Fib from fresh samples due to some analytes could be affected by temperature [10]. Additionally, we collected 438 frozen samples for further analysis of the eight coagulation factors, AT‐III, D‐Di and FDP.
Our study confirmed the concept of developmental hemostasis. Results of our study demonstrated that neonates exhibited distinct coagulation profiles, including prolonged APTT and TT, reduced levels of procoagulant factors (e.g., factor II, IX, X, XI, XII) and anticoagulant factors (e.g. AT‐III), as well as elevated fibrinolytic markers (D‐Di and FDP). Age dependency was most distinct in factor IX, which accords with the result from Appel et al. [15]. The activities of the majority of hemostatic proteins increase with age, and neonates almost always stay at lower levels, consistent with the survey of Di Felice et al [13]. These explained why routine coagulation parameters such as APTT, PT, TT are longer in the neonatal period. Like most studies [4, 6], APTT shows the most significant changes during the growth progress due to its high sensitivity in screening activities of the intrinsic coagulation factors (IX, X, XI and XII), which change greatly during the childhood from low levels to high levels. From data, we could see the activities of coagulation factors IX, XI and XII were low in neonatal period with median as 30.5%, 47.3% and 24.3%, respectively. Besides, the coagulation inhibitor AT‐III, the most important component of the anticoagulant system, remains at a considerably low level in the neonatal period, and then rapidly increases during the infantile period (1‐6 months). Such discrepancies are attributed to the incomplete maturation of the liver in children, resulted in insufficient synthesis of the most hemostatic proteins. As a result, the majority of procoagulant and anticoagulant factors are at relatively low levels at birth, including the vitamin K‐dependent coagulation factors (II, VII, IX, X), contact coagulation factors (XI, XII) and anticoagulant factors (AT‐III, PC, PS) [16].
Levels of D‐Di an FDP were elevated during the whole infancy, particularly the first month of life, and reached adult values by puberty, consistent with the findings of Hudson et al. [17]. and Toulon et al. [4]. Studies pointed out that enhanced activation of coagulation and fibrinolytic system were seen in healthy neonates which might due to the mechanical stress, the adaptation of circulation, and the short‐term hypoxic state during birth [18]. Therefore, high levels of D‐Di and FDP measured during the neonatal period should be interpreted with caution.
Up to now, surveys about the coagulation parameters in Chinese population are still limited and mainly focus on routine coagulation indicators. Large scale analysis for RIs of APTT, PT, TT and Fib were conducted on the STA‐R coagulation analyzer in Zhengzhou, China [7]. Due to the different methodologies and reagent systems used between Sysmex and Stago platforms, lower APTT and PT‐INR reference ranges and patient result were detected on CN‐6000, which can also be observed in our study [19]. Among the Sysmex system, Appel et al. used the Sysmex CA‐1500 to study the RIs of 22 coagulation parameters from 218 European children (aged 1 month to 18 years) from frozen plasma [15]. By comparing the data from our study, we found that coagulation factors (VIII, IX, XII) are much lower in Chinese populations throughout the developmental process. This may be attributed to the ethnic and regional differences as well as the different analyzer and reagent systems used in study. Such data suggested that instrument‐ and age‐specific RIs for the pediatric population are needed.
A newly published survey by Li et al. [20] has established age‐specific RIs for PT, APTT, TT, Fib and coagulation factors (II, V, VII, VIII, IX, X, XI and XII) from 389 children (aged 1 month to 18 years) in Hangzhou, China by the Sysmex CS‐5100 coagulation analyzer. Although minor discrepancies exist in the age stratification between our experimental design and their study, the trends of coagulation‐related parameters examined remain consistent. In our study, we expanded the cohort and stratified participants into six age groups (0–30 days, 1–6 months, 6–12 months, 1–3 years, 3–12 years and 12–18 years) based on the physiological development of children, so as to make more precise estimation of RIs suitable for pediatrics. Besides, we supplemented the data of coagulation function in the neonatal period (0–30 days) which were absent in the above survey, and confirmed that there were significant differences among the RIs for coagulation parameters between the neonates and other age groups. Our data demonstrate the necessity of establishing distinct RIs for coagulation factor IX, as its level exhibits a progressive age‐dependent increase. Moreover, the RIs for fibrinolytic markers (D‐Di and FDP) in Chinese children were also firstly provided in our study.
4.1. Limitations
We acknowledged that the sample sizes of neonates in our study were insufficient and may lead to bias in the estimation of the RIs. This should be considered when interpreting findings. Given the significant difficulty in obtaining samples from healthy newborns, multicenter collaboration is needed for further research. Besides, the division of age groups for pediatric reference intervals, especially for the neonatal period, could make further refinement to gain a deeper understanding of coagulation function in neonates. Meanwhile, our study was conducted at a single tertiary children's hospital in Shenzhen, which may limit the generalizability of RIs to other regions or ethnic groups. Future studies should include diverse geographic and demographic populations. While CLSI EP28‐A3c recommends direct sampling, our use of retrospective data from frozen samples (for AT‐III, D‐Di, FDP) might introduce pre‐analytical variability. Further validation with prospectively collected fresh samples is needed.
5. Conclusions
Coagulation function in children undergoes dynamic evolution with age, and different instruments and reagents can also lead to variations in results. It is necessary for laboratories to establish or validate RIs for children of different ages so as to provide an accurate assessment of coagulation function, screen for bleeding or thrombotic disorders, and monitor the use of anticoagulant and thrombolytic medications among the pediatric population.
Author Contributions
X.Y.F., Y.W. and L.R.B. were responsible for conceptualization, and supervision. Q.Y.D., J.H., D.P., and Y.X.K. were responsible for investigation and validation. L.P.L. was responsible for formal analysis, data curation, and writing – original draft preparation. X.Y.F. and L.R.B. were responsible for writing – review and editing. X.Y.F. was responsible for project administration and funding acquisition. All authors have read and approved the final version of the manuscript. The corresponding author (Xiaoying Fu) had full access to all data and takes responsibility for data integrity and accuracy.
Ethics Statement
The study was approved by the Medical Ethics Committee of Shenzhen Children's Hospital (Approval No. 202501302) and adhered fully to the principles of the Declaration of Helsinki. Written informed consent was obtained from all subjects or patients' guardians involved in the study.
Conflicts of Interest
The authors declare no conflicts of interest.
Transparency Statement
The lead author Ying Wang, Leonardo R. Brandão, Xiaoying Fu affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.
Supporting information
Table S1: Levels of APTT, PT, PT‐INR, TT and Fib expressed as the median and interquartile range (Q1, Q3). Table S2: Levels of D‐Di, FDP, AT‐III and eight coagulation factors (II, V, VII, VIII, IX, X, XI, XII) expressed as the median and interquartile range (Q1, Q3). Table S3: Sex differences in routine coagulation indicators, expressed as median (Q1, Q3).
Acknowledgments
This research was funded by Guangdong High‐level hospital Construction Foundation and Shenzhen Science and Technology Program (No: JCYJ20210324142201004) and Sanming Project of Medicine in Shenzhen (No: SZSM202211033). The funding sources had no role in study design, collection, analysis, and interpretation of data; writing of the report; and the decision to submit the report for publication.
Contributor Information
Ying Wang, Email: 18938690228@163.com.
Leonardo R. Brandão, Email: leonardo.brandao@sickkids.ca.
Xiaoying Fu, Email: xiaoying_fu@foxmail.com.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1: Levels of APTT, PT, PT‐INR, TT and Fib expressed as the median and interquartile range (Q1, Q3). Table S2: Levels of D‐Di, FDP, AT‐III and eight coagulation factors (II, V, VII, VIII, IX, X, XI, XII) expressed as the median and interquartile range (Q1, Q3). Table S3: Sex differences in routine coagulation indicators, expressed as median (Q1, Q3).
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.