Accepted Manuscript

Assessment of two contact activation reagents for the diagnosis ofcongenital factor XI deficiency

Salam Salloum-Asfar, María E. de la Morena-Barrio, JulioEsteban, Antonia Miñano, Cristina Aroca, Vicente Vicente,Vanessa Roldán, Javier Corral

PII: S0049-3848(17)30627-8DOI: https://doi.org/10.1016/j.thromres.2017.12.023Reference: TR 6877

To appear in: Thrombosis Research

Received date: 5 October 2017Revised date: 14 December 2017Accepted date: 29 December 2017

Please cite this article as: Salam Salloum-Asfar, María E. de la Morena-Barrio, JulioEsteban, Antonia Miñano, Cristina Aroca, Vicente Vicente, Vanessa Roldán, Javier Corral, Assessment of two contact activation reagents for the diagnosis of congenital factorXI deficiency. The address for the corresponding author was captured as affiliation forall authors. Please check if appropriate. Tr(2017), https://doi.org/10.1016/j.thromres.2017.12.023

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Assessment of two contact activation reagents for the diagnosis of congenital Factor XI

deficiency

Salam Salloum-Asfar1, María E. de la Morena-Barrio1, Julio Esteban2, Antonia Miñano1, Cristina

Aroca1, Vicente Vicente1, Vanessa Roldán1 and Javier Corral1*.

1Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer,

Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CIBERER, Murcia.

Spain.

2Servicio de Hematología. Hospital Virgen del Castillo de Yecla, Murcia, Spain.

*Contact information for correspondence

Dr. Javier Corral University of Murcia Centro Regional de Hemodonación Calle Ronda de Garay s/n Murcia 30003, Spain Tel: +34968341990/ Fax: +34968261914 E-mail: javier.corral@carm.es

Keywords: FXI deficiency, APTT, silica, ellagic acid, FXI:C, rs1801020.

Highlights

Moderate FXI deficiency might be underestimated by current diagnostic methods.

The sensitivity of two contact activators is evaluatedin140 FXI deficiencycases.

APTT is a low sensitive method for the screening of FXI deficiency.

APTT is strongly dependent on the type of activator and both FXI and FXII levels.

The best screening method forFXI deficiency is FXI:C using silica.

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Abstract

Introduction. Congenital FXI deficiency, a coagulopathy associated with low bleeding risk but

thrombotic protection, is usually diagnosed by prolonged APTT and confirmed by coagulation

assays. Recent evidences suggest that FXI deficiency might be underestimated. Sensitive and

reliable methods to detect FXI deficiency are required. Aim. To examine the sensitivity of two

methods and two contact activators on FXI deficiency screening. Methods. 140 cases with FXI

deficiency, 9 severe and 131 moderate, caused by 11different mutations were recruited. APTT

and FXI:C were assessed in ACL-TOP 500coagulometer with silica-based (SynthASil) and ellagic

acid-based (SynthAFax) reagents. F12 rs1801020 SNP was genotyped with Taqman probes.

Results. Severe FXI deficiency significantly prolonged APTT with both reagents. However, a

high proportion of moderate deficiencies would not be detected using APTT, with false

negatives of 22% for SynthASil and 12% for SynthAFax. False negatives results mainly

corresponded to cases with qualitative deficiency (CRM+: p.Pro538Leu), which also had higher

FXI coagulant activity. Using SynthASil, the common F12 rs1801020 variant, associated to low

FXII levels, significantly prolonged APTT in moderate FXI deficiency subjects. FXI:C values were

significantly higher with SynthAFax than with SynthASil (47.7±12.7 vs. 40.4±14.9), so SynthAFax

rendered higher rate of false negatives than SynthASil (7% vs.2%). Conclusions. Moderate FXI

deficiency, particularly CRM+, might be underestimated using current diagnostic methods. The

activator, FXI and FXII levels may contribute to a higher rate of false negatives using APTT. Our

results suggests that the best screening method for FXI deficiency is FXI:C using silica.

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Introduction

Activated partial thromboplastin time (APTT) is a global coagulation test that has been used

during the last 50 years as a standard screening test in clinical laboratories throughout the

world [1]. APTT levels are considered to reflect global coagulation activity. This test

cumulatively explores factors belonging to the classic intrinsic (FXII, FXI, FIX, and FVIII) or

common (FII, FV and fibrinogen) coagulation cascade. Factor XII is a known determinant of

APTT. Polymorphism rs1801020, which has been associated with FXII levels, showed a highly

significant association with APTT in normal population and case-control cohort of thrombosis

[2, 3]. Besides its sensitivity toward variation levels of these coagulation factors, APTT is also

associated with age, gender, estrogen therapy, and obesity [4].

Generally, coagulopathies involving deficiencies in the intrinsic pathway are evaluated widely

by APTT. Prolonged APTT is an indicator of deficiencies of factors involved the intrinsic

pathway [5, 6]. Numerous reagent systems for performing the APTT assay are commercially

available. However, when the deficiency is mild, these assays vary in their sensitivity to clotting

factor deficiencies.

FXI is a plasma serine protease zymogen of hepatic synthesis with a key role in bridging the

initiation phase and the amplification phase of blood coagulation in vivo [7, 8]. FXI deficiency

originally called haemophilia C, however yields mild bleeding tendency and, in turn, has

significant protective effects from thrombotic diseases [9-11]. FXI deficiency has been

considered a rare disorder and is usually diagnosed by prolonged APTT and confirmed by

coagulation assays (FXI:C<70%). Nevertheless, current reports suggest that this disorder is

present worldwide in many populations and may be underestimated [12-14].

Sensitive and reliable methods to detect FXI deficiency and quantify FXI levels are required. In

this study, using a large cohort of subjects with congenital FXI deficiency caused by 11 different

mutations, we evaluated two assays for their ability to detect deficiencies of FXI, particularly

mild deficiencies. Moreover, two commercially available contact activator reagents were

compared, one based on ellagic acid and one based on silica.

Materials and methods

Study subjects

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The study included 140 subjects (77 male and 63 female donors varying in age from 5 to 88

years) from the Spanish Yecla-study [15], with congenital FXI deficiency characterized

biochemically, functionally and molecularly. Briefly, during 20 years, 324,764 APTT tests from

51,366 hospital in- patients/out-patients were screened, selecting 1,700 patients with an APTT

ratio>1.3 using SynthASil for further studies. Determination of FXI coagulation activity (FXI:C) in

173 patients with replicated and unexplained prolonged APTT (lupus, hepatopathy, and

anticoagulants were excluded) revealed 46 unrelated Caucasian cases with severe to moderate

FXI deficiency (<70% of the value obtained in the reference plasma). Family studies of 265

available relatives from index cases revealed 170 additional subjects with FXI:C deficiency

and/or F11 mutation. The 140 subjects selected for this study belonged to 42 families and

included 9 homozygous or compound heterozygous with severe FXI deficiency and 131

heterozygous with mild-moderate FXI deficiency. FXI deficiency was caused by 11 different

mutations (Table 1).

Table 1. Demographic, analytical and genetic characteristics of the 140 subjects with FXI

deficiency included in this study.

Mutation Ref Mut Type

N (fam)

Genetic Status (N) Age Sex M/F

c.1613C>T; p.Pro538Leu (CM051916)

[13] CRM+ 32 (1)

Homozygous (3) 71.6±5.1 3/0

Heterozygous (29) 34.4±13.7 13/16

c.166T>C; p.Cys56Arg (CM020681)

[16] CRM- 55 (23)

Homozygous &CH (4)

57±10.6 2/2

Heterozygous (51) 41.9±22.1 33/18

c.1247G>A; p.Cys416Tyr (CM053240)

[13] CRM- 18 (7) Heterozygous (18) 50.5±25 9/9

c.1693G>A (CM051917) [17] CRM- 19 (6) Heterozygous (19) 45.5±15.1 8/11

c.1796G>A; p.(Cys599Tyr). New

[15] CRM+ 3 (1) CH (2) 46±2.8 1/1

Heterozygous (1) 81 1/0

c.325G>A (CS081910) [13] CRM- 1 (1) Heterozygous (1) 61 1/0

c.1277T>C; p.(Ile426Thr). New [15] CRM- 4 (1) Heterozygous (4) 47.5±29.9 2/2

c.1608G>C; p.Lys536Asn (CM002953)

[18] CRM- 1 (1) Heterozygous (1) 74 0/1

c.965C>T; p.Thr322Ile (CM950373)

[17] CRM- 1 (1) Heterozygous (1) 44 1/0

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c.802C>T; p.Arg268Cys (CM035499)

[13] CRM- 1 (1) Heterozygous (1) 71 0/1

Exon 8&9 duplication. New [15] CRM- 5(1) Heterozygous (5) 29.4±15.9 3/2

Ref: Reference. Mut Type: Mutation type. CRM-: cross-reacting material-negative. CRM+:

cross-reacting material-positive. M: male; F: female. CH: compound heterozygous

Blood Collection and Plasma Preparation

Blood was collected from the antecubital vein into non siliconized Vacutainer® tubes

containing 3.8% buffered sodium citrate (Becton-Dickinson, Rutherford, NJ). Plasma was

obtained within less than 2 hours after extraction was collected using by centrifugation at

2,500 g for 20 minutes. All plasma samples were aliquoted and stored at -80ºC.

All included subjects gave their informed consent to enter the study performed according to

the declaration of Helsinki, as amended in Edinburgh in 2000. The study was approved by the

Ethical Committee on human research of the Hospital Universitario Reina Sofia in Murcia,

Spain.

Clotting tests

The citrated plasma of all samples was thawed and analyzed the same day. The clotting tests

were performed immediately after thawed, under identical conditions and using the same

batch of reagents for all samples. APTT and FXI:C values were measured using automated ACL

TOP 500 coagulometer (Instrumentation Laboratory) by activating the contact phase with two

different reagents(both from HemosIL): 1) SynthASilTM (colloidal silica-based reagent) and 2)

SynthAFaxTM (ellagic acid-based reagent) using the manufacturer’s standards and calibrators

and following the manufacturer’s instructions. Both reagents used the same CaCl2

concentration (Table 2). For FXI:C clotting assays, FXI deficient plasma (also from HemosIL) was

used following the procedure indicated by the manufacturer.

For APTT, results were expressed either as the raw clotting time in seconds or as a normalized

ratio between the clotting time of the sample and the clotting time of a laboratory reference

standard. A prolonged APTT was considered when the ratio is > 1.3, the cut-off established in

our Hemostasis Unit. For FXI:C clotting activity, results were expressed as % of a laboratory

reference standard.

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Table 2: Features of APTT reagents evaluated in this study

APTT Reagent SynthASil SynthAFax

Part Number and Size 0020006800 APTT reagent 5 x 10 mL 0.020M CaCl2 5 x 10 mL

0020007400 APTT reagent 5 x 10 mL 0.020M CaCl2 5 x 10 mL

Liquid or Lyophilized Liquid Liquid Activator Colloidal Silica Ellagic Acid Phospholipids Synthetic Synthetic Reconstituted Stability at 2-8°C APTT reagent 30 days

in original vial CaCl2 30 days in original vial

APTT reagent 30 days in original vial CaCl2 30 days in original vial

Normal Range * 25.4 - 38.4 seconds 19.7 - 27.6 seconds Heparin Sensitivity +++ ++ Factor Sensitivity +++ ++ Lupus Sensitivity ++ +

*Obtained on the ACL Family of instruments. Source: From HemosIL® - Instrumentation Laboratory- Providing the Right Solutions for Your APTT Testing.

Genetic studies

The polymorphism rs1801020 (c.-4C>T; g.5046C>T) that affects the Kozak sequence of F12 and

has significant functional effects on FXII levels[19] was genotyped by Taqman® probes.

Statistical analysis

Statistical analysis was performed using IBM Statistical Package for Social Sciences (SPSS 21.0;

New York, USA) software. In terms of the management of continuous variables, it was first

verified the normality of its distribution using the Kolmogorov-Smirnov. If the distribution of

data is normal, the majority in our study, is presented as mean ± standard deviation; and are

expressed as median (P25-P75), for non-normal continuous variables. To compare differences

of means among groups, analysis of variance (ANOVA) is used for parametric values, while for

non-parametric values; the U-Mann Whitney tests were applied. Differences resulting from

comparative tests are considered statistically significant when the value of the bilateral "p" is

less than 0.05.

Results

Overall results

Table 3 shows a summary of all the results of APTT and FXI:C obtained in FXI deficiency

patients analyzed by using two activators of the contact phase.

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The silica-based reagent rendered significantly prolonged APTT times but lower APTT ratio

than the ellagic acid-based reagent (Table 3). FXI:C values were significantly lower when

determined with silica than when using ellagic acid.

Severe FXI deficiency (homozygous or compound heterozygous) largely prolonged APTT times

and increased APTT ratios with both silica- and ellagic acid-based reagents (Table 3). This was

also evident for the three homozygous for the CRM+ mutation p.Pro538Leu (Table 3).

The adjustment of the equipment did not allow quantification of FXI:C values in cases with

very severe FXI deficiency (p.Cys56Arg homozygous and compound heterozygous) when

activated by the ellagic acid-based reagent (Table 3).

Table 3.APTT and FXI:C results obtained in 140 patients with FXI deficiency after activation

with two different contact activators: SynthASil and SynthAFax.

FXI Deficiency (N)

APTT seconds (range) SynthASil SynthAFax

APTT ratio (range) SynthASil SynthAFax

FXI:C % (range) SynthASil SynthAFax

All (N= 140)

44.3±13.3 (29.4-115.9)

36.4±16.2 (26.7-132.6)

1.48±0.42 (0.98-3.86)

1.60±0.70 (1.18-5.84)

40.4±14.9 (0.4-73.1)

47.7±12.7 (12.0-83.3)

p < 0.01 < 0.01 < 0.01 Severe deficiency (N= 9)

84.0±25.1 (47.1-115.9)

87.8±34.3 (43.3-132.6)

2.80±0.83 (1.57-3.86)

3.86±1.50 (1.91-5.84)

4.5±5.7 (0.4-12.9)

17.9±1.8 (16.0-19.6)

p 0.86 0.08 0.11 p.Pro538Leu homozygous (N= 3)

52.1±5.3 (47.1-57.6)

46.7±5.2 (43.3-57.2)

1.73±0.17 (1.57-1.92)

2.05±0.22 (1.91-2.32)

12.0±1.3 (10.5-12.9)

17.9±1.8 (16-16.9)

p 0.11 0.11 0.10 Very severe deficiency* (N= 6)

99.9±8.9 (90.4-115.9)

108.3±18.6 (82.2-132.6)

3.33±0.29 (3.01-3.86)

4.77±0.82 (3.62-5.84)

0.7±0.4 (0.4-1.4)

ERROR

p 0.35 0.03 - All heterozygous (N= 131)

41.9±4.4 (29.4-55.1)

32.9±3.5 (26.7-44.7)

1.39±0.14 (0.98-1.84)

1.44±0.15 (1.18-1.97)

42.8±11.8 (11.4-73.1)

48.4±12.0 (12.0-83.3)

p < 0.01 < 0.01 < 0.01 p.Cys56Arg heterozygous (N= 51)

41.7±3.3 (35.2-51.1)

33.2±3.1 (27.1-42.9)

1.38±0.11 (1.17-1.70)

1.46±0.13 (1.19-1.89)

37.9±8.3 (19.7-65.5)

44.1±8.9 (18.3-71.0)

p < 0.01 0.03 0.04 p.Pro538Leu heterozygous (N= 29)

39.8±4.2 (29.4-52.4)

31.1±2.4 (27.6-37.1)

1.32±0.14 (0.98-1.75)

1.36±0.10 (1.22-1.63)

53.7±10.3 (31.7-73.1)

61.2±10.0 (44.0-83.3)

p < 0.01 0.01 < 0.01 c.1693G>A heterozygous (N= 19)

42.8±5.4 (36.9-55.1)

33.4±4.3 (26.7-42.3)

1.42±0.18 (1.23-1.84)

1.45±0.18 (1.18-1.86)

40.5±11.1 (22.0-70.9)

40.9±11.3 (12.0-70.2)

p < 0.01 0.15 0.04

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p.Cys416Tyr heterozygous (N= 18)

43.6±5.2 (35.4-54.3)

34.5±4.6 (29.6-44.7)

1.45±0.17 (1.18-1.81)

1.52±0.20 (1.30-1.97)

42.6±12.0 (23.9-68.8)

46.7±9.8 (27.5-66.7)

p < 0.01 0.02 0.01

* p.Cys56Arg homozygous and compound heterozygous.

Heterozygous subjects with moderate FXI deficiency.

The results of carriers of F11 mutations in heterozygosis has been analyzed globally in the

whole cohort and on the other hand they have been split into four groups of the recurrent

mutations (p.Cys56Arg, p.Pro538Leu, c.1693G>A, and p.Cys416Tyr), which have been analyzed

separately (Table 3). The APTT, expressed both in seconds or as a ratio, was significantly

greater with SynthASil than with SynthAFax for all mutations, except for the APTT ratio for the

mutation affecting the splicing (c.1693G>A). In contrast, for FXI:C, the ellagic acid-based

reagent generated results significantly higher than those generated by silica for all mutations

(Table 3).

Evaluation of sensitivity of SynthASil and SynthAFax reagents for diagnosis of FXI deficiency.

This study was only done for cases with moderate FXI deficiency (heterozygous), as both

methods clearly identify homozygous or compound heterozygous subjects.

Sensitivity refers to a test ability to designate an individual with disease as positive. A highly

sensitive test means that there are few false negative results, and thus fewer cases of disease

are missed. SynthAFax had higher sensitivity (88%) compared to SynthASil (78%) for the

screening of FXI deficiency in subjects with genetic FXI deficiency when using APTT. Thus, the

percentage of false negatives (APTT ratio <1.3) was significantly higher with SynthASil (22.1%)

than with SynthAFax (12.1%) (p<0.01) (Figure 1). When the analysis was done attending to the

mutations, we observed that most (40-46%) of false negatives for both activators

corresponded to cases with qualitative deficiency (CRM+: p.Pro538Leu) (Figure 1).

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Figure 1: Comparison between true positives and false negatives for the APTT ratio depending on whether SynthASil or SynthAFax was used as activator. The percentage of false negatives for heterozygous mutations (p.Cys416Tyr, c.1693G>A, p.Pro538Leu, p.Cys56Arg, p.Ile426Thr, and p.Thr322Ile) is also shown.

Actually, the silica-based reagent rendered higher rates of false negatives (around two times)

than the ellagic acid-based reagent when using APTT ratio for all mutations, except for

c.1693G>A (Table 4).

Table 4: False negatives for the APTT ratio using both different contact activators in

heterozygous carriers of recurrent mutations.

Mutation (N) SynthASil; N (%) SynthAFax; N (%)

p.Cys416Tyr (N= 18) 3 (16.6%) 0 c.1693G>A (N= 19) 4 (21.1%) 4 (21.1%) p.Pro538Leu (N= 32) 13 (40.6%) 8 (25.0%) p.Cys56Arg (N= 51) 9 (17.6%) 4 (7.8%)

Sensitivity was higher for both activators when using FXI:C in heterozygous subjects with FXI

deficiency. However, and in contrast to the results obtained with APTT; the silica-based

activator had higher sensitivity (98%) than the ellagic acid-based activator (93%) (Figure 2).

Thus, the percentage of false negatives observed with SynthAFax was almost three times

higher than with SynthASil (7% vs. 2%, respectively). Again, CRM+ deficiencies are mainly

represented among false negative cases using this assay with both activators (Figure 2).

Figure 2: Comparison between true positives and false negatives for the FXI:C assay

depending on whether SynthASil or SynthAFax was used as activator. The percentage of

false negatives for heterozygous mutations (c.1693G>A, p.Pro538Leu, Exon 8&9 duplication

(dup), and p.Cys56Arg) is also shown.

Indeed, analysis by mutations revealed that only CRM+ mutations have increased rate of false

negatives when evaluating FXI:C levels by using SynthAFax (Table 5).

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Table 5: False negatives for the FXI:C using both different contact activators in heterozygous

carriers of recurrent mutations.

Mutation (N) SynthASil; N (%) SynthAFax; N (%)

p.Cys416Tyr (N= 18) 0 0 c.1693G>A (N= 19) 1 (5.2%) 1 (5.2%) p.Pro538Leu (N= 32) 2 (6.3%) 6 (18.8%) p.Cys56Arg (N= 51) 0 1 (1.9%)

Role of functional F12 polymorphism in the APTT and FXI:C results of subjects with FXI

deficiency.

Since the reagents evaluated in this study activate FXII, variations on the levels of FXII might

significantly affect the results of these assays, particularly APTT. A common F12 polymorphism,

rs1801020 (Minor Allele Frequency –MAF-: 0.472), by disturbing the Kozak sequence of this

gene reduced plasma FXII levels and prolonged APTT results in healthy subjects [19]. We aimed

to evaluate the influence of this polymorphism on APTT and FXI:C assays of subjects with FXI

deficiency. This study was exclusively done in heterozygous carriers since the very low FXI

levels of homozygous or compound heterozygous carriers is the element with biggest weight

on both APTT and FXI:C results.

The rs1801020 polymorphism results in a significantly prolonged APTT in subjects with

heterozygous FXI deficiency only when activated with SynthASil, but it has minor effect when

SynthAFax was used (Table 6). The same effect was observed when we analyzed each mutation

separately, although not significant differences were obtained, probably due to small the

number of patients in each subgroup. As expected, this polymorphism had no effect on FXI: C

values independently of the activator employed (Table 6).

Table 6: Effect of the F12 polymorphism rs1801020 on the APTT and FXI:C values obtained

with SynthASil and SynthAfax from subjects with heterozygous FXI deficiency

SynthASil SynthAFax

Mutation

(N) Genotype rs1801020

N APTT (sec) APTT (ratio)

FXI:C (%) APTT (sec) APTT (ratio)

FXI:C (%)

All cases (N= 131)

CC CT/TT

88 43

41.2±4.3 42.4±7.8

1.37±0.14 1.44±0.14

43.0±12.7 42.4±9.5

32.7±3.4 32.4±6.3

1.44±0.15 1.46±0.16

48.7±13.1 47.9±9.4

p 0.69 0.04 0.77 0.88 0.59 0.95

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p.Cys56Arg (N= 51)

CC CT/TT

36 15

40.9±3.2 41.5±11.9

1.36±0.11 1.47±0.15

40.5±10.6 39.9±9.9

32.8±2.8 32.1±9.5

1.44±0.12 1.51±0.19

47.0±11.2 44.9±9.9

p 0.22 0.13 0.95 0.44 0.36 0.46

p.Pro538Leu (N= 29)

CC CT/TT

23 6

38.8±3.3 41.9±2.5

1.29±0.11 1.39±0.09

49.5±11.8 48.2±6.4

30.9±2.3 30.5±0.9

1.36±0.10 1.34±0.04

55.6±13.0 55.9±9.8

p 0.33 0.33 0.93 0.95 0.76 1.00

c.1693G>A (N= 19)

CC CT/TT

11 8

41.4±5.1 43.2±5.5

1.38±0.17 1.44±0.18

44.7±15.2 42.2±11.1

32.2±4.6 33.2±4.2

1.42±0.20 1.46±0.18

44.6±20.0 46.3±9.7

p 0.93 0.61 0.61 0.74 0.51 0.51

p.Cys416Tyr (N= 18)

CC CT/TT

8 10

45.4±5.2 43.3±3.5

1.51±0.18 1.44±0.12

38.5±13.5 44.4±8.7

36.4±3.8 33.0±3.1

1.60±0.17 1.46±0.14

43.7±9.5 51.1±6.9

p 0.82 0.94 0.17 0.14 0.55 0.04

Role of age and gender on the APTT results of subjects with FXI deficiency.

In our cohort, gender has not a significant effect on APTT of all carriers of any heterozygous

mutation, however when we analyzed each recurrent mutation separately, we found that

women have significant lower values of APTT in carriers of c.1693G>A mutation (Table 7).

Table 7: Effect of gender on the APTT obtained with SynthASil and SynthAfax from subjects with heterozygous FXI deficiency and in each recurrent mutation. SynthASil SynthAFax

Mutation (N) Male (71) Female (60) Male (71) Female (60) All heterozygous (N= 131)

APTT (sec) p APTT ratio p

41.7±6.5 0.38

1.41±0.14 0.31

41.4±4.6 1.38±0.15

32.4±5.2 0.73

1.45±0.16 0.36

32.7±3.4 1.44±0.15

Male (33) Female (18) Male (33) Female (18) p.Cys56Arg heterozygous (N= 51)

APTT (sec) p APTT ratio p

40.5±7.9 0.73

1.39±0.11 0.60

42.3±4.8 1.41±0.16

31.8±6.4 0.50

1.44±0.13 0.52

34.0±3.9 1.49±0.17

Male (13) Female (16) Male (13) Female (16) p.Pro538Leu heterozygous (N= 29)

APTT (sec) p APTT ratio p

40.1±2.6 0.48

1.34±0.08 0.48

38.4±3.7 1.28±0.12

31.2±2.4 0.78

1.37±0.11 0.78

30.6±2.0 1.35±0.09

Male (11) Female (8) Male (11) Female (8) c.1693G>A heterozygous (N= 19)

APTT (sec) p APTT ratio p

44.0±7.1 0.01

1.47±0.24 0.01

41.3±3.2 1.37±0.11

34.1±5.8 0.02

1.50±0.25 0.01

31.8±0.26 1.40±0.11

Male (9) Female (9) Male (9) Female (9) p.Cys416Tyr heterozygous (N= 18)

APTT (sec) p APTT ratio p

46.3±4.9 0.09

1.54±0.16 0.09

42.2±2.7 1.41±0.09

35.4±4.4 0.44

1.56±0.19 0.46

33.6±2.9 1.48±0.13

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Regarding age, no significant effect observed on the APTT, independently of the activator

(Table 8). No subjects below 15 years old carry c.1693G>A or p.Cys416Tyr mutations.

Table 8: Effect of age on the APTT obtained with SynthASil and SynthAFax from subjects with heterozygous FXI deficiency and in each recurrent mutation.

SynthASil SynthAFax

Mutation (N) Age <15 (10) Age >15 (121) Age <15 (10) Age >15 (121) All heterozygous (N= 131)

APTT (sec) P APTT ratio P

40.7±3.2 0.67

1.36±0.11 0.81

41.7±5.9 1.40±0.15

31.9±2.48 0.58

1.41±0.11 0.57

32.6±2.6 1.45±0.16

Age <15 (5) Age >15 (47) Age <15 (4) Age >15 (47) p.Cys56Arg heterozygous (N= 51)

APTT (sec) P APTT ratio P

40.4±4.6 0.50

1.34±0.16 0.52

41.2±7.2 1.40±0.13

32.3±3.4 0.83

1.42±0.15 0.85

32.6±5.9 1.46±0.15

Age <15 (5) Age >15 (25) Age <15 (5) Age >15 (25) p.Pro538Leu heterozygous (N= 29)

APTT (sec) P APTT ratio P

39.7±1.8 0.26

10.32±0.06 0.26

39.1±3.5 1.30±0.11

30.9±1.6 0.25

1.36±0.07 0.25

30.9±2.3 1.36±0.10

Discussion

FXI deficiency has been considered a rare disease except for some specific populations. Thus

among Ashkenazy Jews, FXI deficiency may be present in up to 8% of the population [8, 20].

However, a recent study evaluating available whole exome sequencing from different

populations found a frequency higher than expected of pathogenic mutations leading to FXI

deficiency [21]. This apparent contradiction may be explained by two reasons: 1) current

diagnostic methods for FXI screening fail to identify subjects with FXI deficiency, particularly

with mild FXI deficiency; 2) the clinical consequences of FXI deficiency (particularly bleeding)

are very mild or absent. Our data shows new evidences on the strong limitation of APTT as a

screening method for the diagnosis of FXI deficiency. Although all severe FXI deficiencies

significantly prolonged APTT using any activator, a high proportion of cases with mild-

moderate FXI deficiencies had APTT values within the normal range and may be missed. The

rate of false negatives is particularly high when using the silica-based reagent. This method

particularly fails to detect a CRM+ deficiency, p.Pro538Leu, as almost half of heterozygous

carriers of this mutation (40.6%) had APTT ratios within the normal range (< 1.3). This

observation may be explained by the strong dependence of APTT on FXI coagulant activity

(Figure 3) and the higher coagulant activity of FXI among heterozygous carriers of the

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p.Pro538Leu(53.7±10.3withSynthASil and 61.2±10.0withSynthAFax)compared with carriers or

all other mutations (40.25±12.46 with SynthASil and 45.90±10.85 with SynthAFax).

Figure 3: Percentage of false negatives of APTT ratio with FXI:C levels.

Thus, moderate levels of FXI:C (>50%) are correlated with high rate of false negatives,

especially with SynthASil reaching 41% (Figure 3).

Our study shows some other clues that contribute to explain the low specificity of this method.

The activator of the contact phase is really important. Thus, the rate of false-negatives

increases two-fold (although it is still quite high) when using a stronger activator (the ellagic

acid reagent in our study). Moreover, the common and functional rs1801020SNP affecting the

plasma levels of FXII also contributes to reduce the sensitivity of APTT to detect moderate FXI

deficiency. Subjects with moderate FXI deficiency that also have high levels of FXII in plasma

(carriers of the C/C genotype of rs1801020 SNP, which represent up to 65% of the general

Spanish population[19]) had more possibilities to have normal APTT values. On the other hand,

it has been previously described that APTT were significantly lower in women than in men in

random population[4].In our cohort, gender has not a significant effect in all carriers of any

heterozygous mutation, however when we analyze each recurrent mutation separately, we

found that women have significant lower APTT ratios in carriers of c.1693G>A mutation. These

data support that APTT is neither a specific nor sensitive method to diagnose mild FXI

deficiency[22] and interference of other factors such type of activator, F12 polymorphism,

FXI:C levels and gender may lead to false interpretation of APTT values.

Our study shows that FXI:C assays have much better sensitivity to diagnose FXI deficiency.

Moreover, this method is not influenced by any of the studied factors (age, gender, or F12

0%

19%

41%

0% 4%

27%

0%

50%

100%

<20 20-50 >50

% o

f fa

lse

ne

gati

ves

of

AP

TT r

atio

% of FXI:C

SynthASil SynthFAx

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SNP), and the activator has a mild effect, as although the rate of false negatives is almost three

times lower with SynthASil, than with SynthAFax, the rates are small (2% vs. 7%, respectively).

Our study has some strengths: 1) it has been done with one of the largest series of patients

with FXI deficiency collected worldwide, particularly of Caucasian origin (N= 140); 2) the study

has not been restricted to carriers of a single molecular defect, although there is a noticeably

molecular homogeneity as the majority of cases are heterozygous carriers of 4 recurrent

mutations identified in our population; 3) the study had been performed on the same day,

using the same reagents, same controls and the same coagulometer, reducing then

experimental variations; 4) we have evaluated two commercial contact activator reagents used

for the two routinely tests used for the diagnosis of FXI deficiency: APTT and FXI:C. However,

our study also has some limitations. It has been done with selected cases with FXI deficiency,

some of them selected by having a prolonged APTT using SynthASil. Moreover, our study has

been restricted to available carriers of described mutations. Finally, we have only evaluated

two contact activators reagents from a single company. Although further studies are required

to validate these results and to study other contact activators, our data suggests that the best

way to diagnose a case with FXI deficiency is by FXI:C using a weak activator (SynthASil in our

case).

Conclusions

Moderate FXI deficiency, particularly CRM+, might be underestimated by the limitations of

current diagnostic methods. APTT has particularly low sensitivity for FXI diagnosis as it is

influenced by FXII levels (determined by a common F12 SNP), by FXI:C levels and by the

activator of the contact phase. Our results suggests that the best screening method for FXI

deficiency is FXI:C using silica.

Funding

This work was supported by PI15/00079; CB15/00055 (ISCIII and FEDER); and 19873/GERM/15

(Fundación Séneca). SS-A is the recipient of the Research Fellowship from the Spanish Society

of Hematology and Hemotherapy (SEHH).MEM holds a fellowship from Fundación Española de

Trombosis y Hemostasia (FETH).

Acknowledgments

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This research was supported by Izasa Scientific/A WerfenLife Company. The authors are

thankful to Alberto Lopez and Josep Garcia for assistance and the provided expertise that

greatly assisted the research.

Authorship

S.S-A., J.E., M.E.M-B., J.C., V.R. and V.V. collected patients and clinical data, designed research,

analyzed the data and wrote the paper. A.M. and C.A. performed genetic and biochemical

experiments.

Conflict of interest

Authors have no conflict of interest.

References

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