Longer aPTT Values in Healthy Children than in Adults: No Single Cause

THROMBOSIS RESEARCH

Pergamon

Thrombosis Research 88 (1997) 355-359

REGULAR ARTICLE

Longer aPTT Valuesin Healthy Childrenthan in Adults:No SingleCause Siegfried Gallistl’, Wolfgang Muntean’, Bettina Leschnik’ and Wilfried Meyersz ‘Department of Pediatrics, University of Graz, Austria and 2Behringwerke, Marburg, Germany (Received 25 April 1997 by Editor L Pabinger; revised/accepted 20 September 1997)

Abstract We have shown that activated partial thromboplastin time values in children are considerably longer than in adults, but the causes for this observation remained unclear. Therefore, we investigated the correlation between activated partial thromboplastin time values and concentrations of clotting factors, quotients and titers of the tissue thromboplastin inhibition test, and antiphospholipid antibodies in healthy children, children with recurrent infections, and adults. Concentrations of factors VIII, IX, and HMWK were significantly lower in children than in adults. Simple linear regression analysisfailed to show a correlation between the concentration of a single clotting factor and the activated partial thromboplastin time values. No significant correlation was found between activated partial thromboplastin time and elevation of the tissue thromboplastin inhibition test quotients or titers, or antiphospholipid antibodies values. The determined activated partial thromboplastin time was best described by a function including all measured coagulation factors. Our study suggests, that no single clotting factor or lupus anticoagulants are responsible for the longer activated partial thromboplastin time in healthy children, but that actiAbbreviations: aPTT, activated partial thromboplastin time; ITIT, tissue thromboplastin inhibition test; APA, antiphospholipid antibodies; LA, lupus anticoagulants; HMWK, high molecular weight kininogen; PK, prekallikreine. Corre~ponding aathor: Dr. Siegfried Gallistl, Univ. Klinik fur Kinder u. Jugendheilkunde, Auenbruggerplatz 30, A- 8036 Graz, Austria. Tel: (+43) 316385 2605; Fax: (+43) 3163853300.

vated partial thromboplastin prolongation is caused by the combination of several slightlylower clotting factors. @1998 Elsevier Science Ltd. Key Words: aPTT; Children; Lupus anticoagulants; Antiphospholipid antibodies

T

he activated partial thromboplastin time (aPTT) is the most widely used screening test to detect hereditary and acquired coagulation defects. In a previous study we have shown that in about 50Y0of healthy children the aPTT values are over the 95th percentile of the aPTT values of healthy adults [1]. This observation is in accordance with the clinical experience when testing healthy children preoperatively, if there is no obvious explanation for a prolonged aPTT in children: in neonates it has been shown that longer aPTT values are due to physiologic low levels of contact factors and factor IX [2–6]. Although plasma concentrations of many coagulation proteins reach adult ranges by 6 months of age, the mean values of several coagulant are significantly lower in healthy children than in adults [7]. However, in healthy children no single clotting factor is low enough to sufficiently explain prolongation of the aPTT, which possibly may be also caused by lupus anticoagulants (LA) [8]. LA, members of the antiphospholipid antibodies (APA) family, not only appear in autoimmune disorders or lymphoproliferative diseases [9], in many cases LA are a transient laboratory finding in the setting of convalescence from recent infections [10].Since children repeatedly suffer from minor viral infections LA

0049-3848/97$17.00 + .00 01998 Elsevier Science Ltd. Printed in the USA. All rights reserved. PII SO049-3848(97)O0265-X

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S. Gallistl et al./Thrombosis Research 88 (1997) 355-359

1. Patients and Methods

(PK) were determined by one-step methods using deficient plasmas @ehring, Marburg, FR@ LA were searched for using a modified tissue thromboplastin inhibition test (TTIT) [15]: reference and patient plasma were incubated with 1:100diluted human tissue facta--based thromboplastin (Thromborel, Behring, FRG). Activation was started by the addition of 0.025 M CaC12,and clotting times were compared.. In the case of prolonged clotting time of patient plasma, reference plasma was added until clotting time normalized. This test was assessed as follows: 1) TTIT ratio, as”ratioof clotting time of undiluted patient plasma to clotting time of reference plasma; and 2) TTIT titer as titer of normalization of the diluted patient plasma. APA were determined using ELISA technique (Boehringer, Mannheim, FRG).

1.1. Subjects

1.3. Statistical Analysis

Fifty-six healthy children (age: 1–16 years; mean: 7.5 years; 31 male, 25 female), 54 children with recurrent pharyngitis (age: 1.5–15.5years; mean: 7.2years; 28 male, 26 female), and 54 healthy adults receiving no medications (age: 22–55years; mean: 33.3years; 32 male, 22 female) were eligiblefor this study. Healthy children and children with recurrent pharyngitis were seen for preoperative examination prior to minor surgery or tonsillectomy/adenoidectomy, respectively. Most adults were members of the staff. To qualify for the study, the following criteria had to be met: no family history of bleeding and thrombophilia, no history of easy bruising, a normal physical examination and a normal white blood cell count.

The significanceof differences between the groups was determined with the Wilcoxon’s test. Simple linear regression.was used to assess the correlation between concentrations of clotting factors, TTIT ratios and titers, APA, and the aPTT. P values less than 0.05were considered to represent a significant difference or relationship.

might be frequently observed in otherwise healthy children. LA are immunoglobulins which interfere with phospholipid dependent coagulation tests, thereby leading to clotting time. prolongation [11,12].In addition, it has been shown that LA are frequently associated with low factor XII ,levels [13]and that patients with LA and thrombosis have a higher prevalence of low factor XII than the general population [14].The aim of this study was to investigate whether the longer aPTT in children might be due to tower concentrations of clotting factors, or the presence of LA that may be more often transiently present in children.

1.2. Laboratory Nine parts of blood were drawn, using a two syringe technique, from an antecubital vein into syringes containing one part of 0.1 M citrate at the same time that routine preoperative complete blood work was obtained. Blood was centrifuged at 1700 g, plasma was removed and immediately used for coagulation tests. Plasma for APA (anticardiolipin antibodies) ELISA was stored at –70”C. aPTT was measured by a clotting method using a kaolin containing reagent (Pathrombtin, Behring, Marburg, FRG). Factors VIII, IX, XI, XII, high-molecular weight kininogen (HMWK), and prekallikreine

2. Results Mean values and SD for each of the tests performed are presented in Table 1, APTT values in healthy children (range: 33.3–56.0 seconds) and children with recurrent infections (range: 33.5–56.1 seconds) were significantly longer than in adults (range: 28.5-48.0 seconds). Forty-one percent of healthy children and 547. of children with recurrent infections showed aPTT levels longer than the 95th percentile of healthy adults (40.8 seconds). Two healthy children and three children with recurrent infections showed markedly prolonged aPTT values (10 seconds above the 95th percentile of healthy adults). Four of these children showed TTIT titers higher than 1:10.Factors VIII, IX, and HMWK were significantly lower in healthy children and children with recurrent infections than in the adult group. Comparing healthy children and children with recurrent infections, factors VIII and XIrwere lower in children with recurrent infections

S. Gallistl et al./Thrombosis Research 88 (1997) 355-359

357

Table 1. Results of the investigated parameters in adults, healthy children. and children with recurrent infections

aPTT (see) VIII (7.) IX (Ye) XI (7.) XII (%) HMWK (%) PK (Ye) TTIT ratio APA (U/1)

Adults

Healthy children

Children with recurrent infections

35.2 (3.2) 152 (48) 116 (30) 108 (27) 121 (48) 105 (28) 107 (29) 1.0 (0.03) 5.03 (3.4)

40.6 (4.3)’ 136 (51)’ 97 (30)’ 106 (25) 122 (35) 85 (19)’ 109 (31) 1.03 (0.03)’ 6.03 (4.4)

42.0 (5)’ 114 (38)ab 94 (31)’ 97 (24) 104 (43)’JJ 88 (20)’ 110 (27) 1.05 (0.05)’ 5.05 (3.43)

Data are presented as meanslSD. aValues are significantly different from adults. bValues are significantly different from healthy children.

than in healthy children. There was no difference in concentration of APA between all groups. The TTIT ratio was significantlyhigher in healthy children and children with recurrent infections than in adults. As can be seen in Figure 1, 28Y0of adults (versus 4% of healthy children) did not show a prolonged clotting time in the TTIT compared with reference plasma. In addition, 65Y0of adults (versus 32~0 of healthy children) normalized when plasma was diluted 1:2 with reference plasma. However, when patient plasma was diluted up to 1:5 with reference plasma, 95?40of adults and healthy children showed normalization of clotting times in the TTIT. In contrast, only 72Y0of children with recurrent infections normalized up to a titer of

1:5 (p< O.001).Children with recurrent infections, who normalized at titers higher than 1:5 showed significant longer aPTT values than healthy children (p< O.01). 2.1. Correlation between Coagulation Factors, TTIT, APA, and aPPT Values Using simplelinear regression we found no correlation between the concentration of a single clotting factor and the determined aPTT. In addition, comparison of TTIT ratio, TTIT titers, or concentrations of APA failed to show a significant correlation with the aPTT. There was not correlation between the concentration of APA and the results

100, ❑ healthy children 90

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■ children w. rec. infect 80 70

Fig. 1. Titers of normalization in the TTIT (TTIT titer) in healthy children, healthy adults, and children with recurrent infections (children w. rec. infect.)

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S. Gallistl et al./Thrombosis Research 88 (1997) 355-359

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of the T’TIT.The determined aPTT in each of the investigated groups was best described by a function that included all measured coagulation factors: calculated aPTT=50.5 sec–O.032 [VIII]– 0.033 IIX]–O.026 IXI]+O.005 [XII]+ 0.027 [PK]–O.053 [HMWK].

Figure 2 shows the significantcorrelation between calculated aPTT and actually measured aPTT. 3. Discussion Our present results confirm our earlier study showing that aPTT values in children are considerably longer than in adults [1].A convincingexplanation for this observation had been missing.Prolongation of the aPTT is commonly due to decreased levels of the contact factors (XI, XII, PK, HMWK), and factors VIII and IX. In addition, LA may prolong phospholipid dependent tests of coagulation by binding to phospholipid surfaces, thus preventing assembly of enzymatic coagulation complexes [12]. Recently, we demonstrated a higher frequency of decreased factor XII concentration in children with elevated APA or positive TTIT [16], which can be observed after a variety of infections including common viral infections [17,18].Again, in the pres-

ent study we found a significant higher frequency of elevated TTIT titers and decreased factor XII levels in children with recurrent pharyngeal infections. These children showed longer aPTT values than healthy children and adults. However, in the entire groups neither the concentration of factor XII, nor the TTIT ratio or the TTIT titer correlated with the determined aPTT. Interestingly, 65Y0of adults showed normalization in the TTIT at a titer of 1:2 compared to only 32Y0of healthy children (Figure 1), though the frequency of elevated TTIT titers and APA was nearly the same in both groups. Prolongation of TTIT is not specific for LA [8]. Since LA and APA usually appear coincidental in approximately 60Y0of patients [11], the lack of correlation between TTIT values and APA in each of the investigated groups argues against the presence of LA as a frequent cause of aPTT prolongation in healthy children. Although healthy children and children with recurrent infections showed significant lower levels of factors VIII, IX, and HMWK compared to adults, we could not find a correlation between one of these coagulation factors and the determined aPTT. The measured aPTT values were best described by a function consisting of factors VIII, IX, XI, XII, HMWK, and PK. As shown in Figure 2, this function can be applied to each of the investigated groups and

S. Gallistl et al.tThrombosis Research 88 (1997) 355-359

gives a significant correlation with the actually determined aPTT values. It has been shown that aPTT reagents vary in their sensitivityto LA, both in terms of overall sensitivity and in sensitivity to specific LA [19]. The kaolin containing aPTT reagent used in our study is sensitive to LA. Eberl et al. demonstrated aPTT normalization using an inhibitor insensitive aPTT reagent in children with prolonged aPTT and evidence for coagulation inhibitors [20].Reagent dependent different sensitivities must be taken into consideration when normal data of a distinctpopulation are evaluated. Andrew et al. found decreased factors II, V, IX, X, XI, and XII in healthy children aged 11 to 16 years compared to adults, but no difference concerning the aPTT using Actin FS [7]. Thus, comparison of data obtained using different reagents might not be reliable. However, our study suggests, that neither a single clotting factor nor LA are responsible for the longer aPTT values in healthy children, but that aPTT prolongation is caused by the combination of lower concentrations of several clotting factors. References 1. Muntean W, Finding K, Leschnik B. Partial

2.

3.

4.

5. 6. 7.

8.

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