Newborn and Pediatric Reference Intervals for Coagulation Assays Using Novel Reagents

Age-specific differences in the concentration and function of several hemostatic proteins are well established and are encompassed by the concept of developmental hemostasis.[1] [2] [3] [4] The functional measurement of hemostatic proteins is also reagent- and analyzer-specific.[3] [5]

Despite, coagulation assays such as fibrinogen and prothrombin time (PT) being long-standing assays, the reagents used, specifically the ones from Diagnostica Stago, France are constantly being modified to be compliant with regulatory guidelines to ensure a residual activity of <1% (e.g., STA-ImmunoDef VIII and ImmunoDef IX) and optimized to achieve an ideal international sensitivity index (e.g., STA-NeoPTimal). Improvements have also been made in reagents used to measure fibrinogen (e.g., STA-Liquid-fib), with an increase in the amount of titrated human calcium thrombin.

With the continual development of new reagents and analyzers, the previously established age-specific reference intervals become outdated, leading to the need for establishment of new reference intervals, to ensure the accurate interpretation of results.

This study aimed to establish pediatric reference intervals for activated partial thromboplastin time (APTT), PT, fibrinogen, D-dimer, fibrin degradation products (FDP), factor VIII (FVIII), and factor IX (FIX) using novel Stago Reagents (Diagnostica Stago, France), not used previously,[4] on the STA R Max analyzer (Diagnostica Stago, France).

Venous blood samples were collected from healthy neonates and children without previous thromboembolic events, past or family history of bleeding, and who were not subjected to any form of anticoagulant therapy, according to a previously established protocol.[6] This study was approved by the Royal Children's Hospital, Human Research Ethics Committee (HREC #34183) and the Royal Women's Hospital Human Research Ethics Committee (#2/08). Written informed consent was obtained from parents/guardians of the children.

Blood samples were collected in 3.2% sodium citrate S-Monovette tubes (Sarstedt, Australia), and were centrifuged at 3,000 rpm for 10 minutes at room temperature (Megafuge 1.0R, Heraeus), with platelet-poor plasma stored at −80°C until testing. Frozen plasma samples were thawed at 37°C in a water bath and were homogenized by gentle inversion. Samples were then tested within 2 hours of thawing.

APTT, PT, fibrinogen, D-dimer, FDP, FVIII, and FIX were measured using the following reagents: C.K. Prest, NeoPTimal, Liquid-Fib, Liatest D-Di Plus, Liatest FDP, ImmunoDef VIII, and ImmunoDef IX, respectively, on an automated STA Max analyzer (Diagnostica Stago, France).

Results were presented in the following age groups: newborns (from birth to 1 month of age), infants (greater than 1 month to 3 years of age), children (greater than 3 to 13 years of age), and adolescents (greater than 13 to 18 years of age). Data analysis was performed using GraphPad Prism (version 9.0) and results are expressed as mean with intervals including 95% of the population (2.5th–97.5th percentile) in accordance with the Clinical and Laboratory Standards Institute guidelines.[7] Results from the newborn and pediatric age groups were compared with the adolescents using a one-way ANOVA (analysis of variance), followed by Dunnett's test to correct for multiple comparisons. A p-value of <0.05 was considered to be statistically significant.

APTT, PT, fibrinogen, D-dimer, FDP, FVIII, and FIX levels were assessed in a total of 520 participants with participant characteristics described in [Table 1]. The respective reference intervals are shown in [Table 2].

Table 1 Participant demographic data

	Newborns (birth to 1 mo)	Infants (>1 mo to 3 y)	Children (>3 to 13 y)	Adolescents (>13 to 18 y)
Participants (n =)	134	123	141	122
Median age	2 d	2.0 y	6.9 y	14.7 y
Age range	0–29 d	0.3–2.9 y	3.0–12.9 y	13.1–17.9 y
Sex (M/F)	64/70	75/48	79/62	64/58

Table 2 Mean values with age-specific reference intervals for activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, D-dimer, fibrin degradation products (FDP), factor VIII and factor IX

	Newborns (birth to 1 mo)	Infants (>1 mo to 3 y)	Children (>3 to 13 y)	Adolescents (>13 to 18 y)
APTT (s)
n = Mean Reference interval	40 38.1[a] 29.9–52.8	41 33.2 28.2–42.3	44 32.0 26.8–36.0	41 31.2 26.2–38.6
PT (s)
n = Mean Reference interval	44 17.6[a] 12.8–24.9	45 14.1 12.6–16.6	43 14.7 13.3–16.8	43 14.6 12.8–15.8
Fibrinogen
(g/L) n = Mean Reference interval	44 3.1[a] 1.9–4.8	45 2.5 1.8–3.3	43 2.7 2.0–3.8	43 2.7 1.8–3.5
D-dimer (µg/mL FEU)
n = Mean Reference interval	44 1.5[a] 0.3–3.9	43 0.3 0.3–0.6	44 0.3 0.3–0.7	42 0.3 0.3–0.4
FDP (µg/mL)
n = Mean Reference interval	44 6.5[a] 4.0–21.0	43 4.0 4.0–5.3	44 4.0 4.0–4.0	42 4.0 4.0–4.0
Factor VIII (%)
n = Mean Reference interval	40 152.1 50.1–283.0	41 128.4 68.7–247.9	44 133.5 79.2–303.4	41 147.4 72.5–325.6
Factor IX (%)
n = Mean Reference interval	40 72.7[a] 40.1–107.4	41 90.6[a] 65.5–124.2	44 96.9[a] 75.2–147.8	41 116.4 48.8–180.7

^a p-Value significantly different from adolescents (p < 0.05).

The findings of this study are comparable with previously published works, with age-specific differences between newborns and adolescents across several functional assays such as APTT, PT, D-dimer, and FIX, with the exception of FVIII levels.[3] [4] Previous pediatric reference interval studies have reported that fibrinogen levels among newborns do not differ significantly when compared with adolescents.[4] Conversely, we have shown that there are age-specific differences in fibrinogen levels among newborns when compared with adolescents. These inter-study differences could be due to the differences in Stago reagents utilized, namely the novel STA-liquid-fib and outdated STA-fibrinogen kit (Diagnostica Stago, France). Furthermore, the age categories were not consistent across studies, which could result in the differences seen. Monagle et al investigated the age-specific differences in neonates (day 1 and 3 of life), whereas this study investigated the differences in newborns (birth to 1 month old).

In addition, differences in absolute reference interval values were also seen across all functional assays, when compared with previously published work.[3] [4] [8] [9] [10] Such discrepancies among reference interval values are likely due to the difference in age categories, reagents as well as analyzers used across studies.[3] [4] [5] This is important as disease states are considered when patients' results are outside of the 95th centile, and so even if the means of parameters are not statistically different, these results provide important information for diagnosis of children by giving ranges that can be considered normal for comparison when these new reagents are used clinically.

In conclusion, there are significant differences within pediatric reference intervals from newborns to adolescents, highlighting the need for reagent and analyzer-specific reference intervals across the age spectrum, thus enabling accurate interpretation of coagulation tests and accurate diagnosis for newborns and children.

Publikationsverlauf

Eingereicht: 23. März 2022

Angenommen: 06. Juli 2022

Artikel online veröffentlicht:
07. November 2022

• References

• 1 Andrew M, Paes B, Milner R. et al. Development of the human coagulation system in the healthy premature infant. Blood 1988; 72 (05) 1651-1657
• 2 Andrew M, Vegh P, Johnston M, Bowker J, Ofosu F, Mitchell L. Maturation of the hemostatic system during childhood. Blood 1992; 80 (08) 1998-2005
• 3 Attard C, van der Straaten T, Karlaftis V, Monagle P, Ignjatovic V. Developmental hemostasis: age-specific differences in the levels of hemostatic proteins. J Thromb Haemost 2013; 11 (10) 1850-1854
• 4 Monagle P, Barnes C, Ignjatovic V. et al. Developmental haemostasis. Impact for clinical haemostasis laboratories. Thromb Haemost 2006; 95 (02) 362-372
• 5 Mackie I, Cooper P, Lawrie A, Kitchen S, Gray E, Laffan M. British Committee for Standards in Haematology. Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis. Int J Lab Hematol 2013; 35 (01) 1-13
• 6 Hoq M, Karlaftis V, Mathews S. et al. A prospective, cross-sectional study to establish age-specific reference intervals for neonates and children in the setting of clinical biochemistry, immunology and haematology: the HAPPI Kids study protocol. BMJ Open 2019; 9 (04) e025897
• 7 Clinical and Laboratory Standards Institute. ed. Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory: Approved Guideline. 3. ed. Clinical and Laboratory Standards Institute; 2010
• 8 Appel IM, Grimminck B, Geerts J, Stigter R, Cnossen MH, Beishuizen A. Age dependency of coagulation parameters during childhood and puberty. J Thromb Haemost 2012; 10 (11) 2254-2263
• 9 Toulon P, Berruyer M, Brionne-François M. et al. Age dependency for coagulation parameters in paediatric populations. Results of a multicentre study aimed at defining the age-specific reference ranges. Thromb Haemost 2016; 116 (01) 9-16
• 10 Flanders MM, Crist RA, Roberts WL, Rodgers GM. Pediatric reference intervals for seven common coagulation assays. Clin Chem 2005; 51 (09) 1738-1742