Haemophilia (2014), 1–3
DOI: 10.1111/hae.12585
LETTER TO THE EDITOR
Bleeding phenotype in carriers of haemophilia A does not correlate with thrombin generation € ¶ and F . B A G H A E I* A . O L S S O N , * M . H E L L G R E N , † ‡ E . B E R N T O R P , § M . H O L M S T R OM *Department of Haematology and Coagulation Disorders, Sahlgrenska University Hospital, Gothenburg; †Department of Antenatal Care N€ arh€ alsan Primary Care, V€ astra G€ otaland; ‡Department of Obstetrics and Gynaecology Institute of Clinical Science, Sahlgrenska Academy, University of Gothenburg, Gothenburg; §Centre for Thrombosis and Haemostasis Sk ane University Hospital, Lund University, Malm€ o; and ¶Coagulation Unit Haematology Centre, Karolinska University Hospital, Stockholm, Sweden
Haemophilia A is an X-linked disorder. Females who inherit the mutation are known as haemophilia A carriers. Clotting factor level in carriers are typically about 50% of levels in the normal population, but may vary considerably due to random X inactivation. Studies have reported increased bleeding tendency among carriers, especially in situations that challenge haemostasis, such as surgery [1]. In our previous study [2], we found an increased bleeding tendency in a cohort of carriers of moderate and severe haemophilia. Bleeding tendency was weakly correlated with clotting factor levels in haemophilia A carriers, but there was nonetheless a group of carriers with increased bleeding tendency despite normal levels. The reverse condition, a normal bleeding tendency combined with lowered factor levels, also existed in a subgroup of the carriers. This suggests that clotting factor levels alone may not be sufficient to reflect or predict bleeding tendency among carriers. Thrombin is a key enzyme in the coagulation process and thrombin generation assays (TGA) are used as a measure of global haemostasis. The thrombin generation profile of haemophilia A has been characterized [3,4] and TGA has illustrated the clinical heterogeneity among individuals with haemophilia with the same clotting factor levels [5]. Since clotting factor levels did not adequately predict bleeding risk in our cohort of carriers, we wanted to investigate whether measuring overall haemostasis with TGA might be more appropriate. The primary aim of this study was to compare TGA results in carriers of moderate and severe haemophilia A with increased bleeding tendency with those in carriers without increased bleeding tendency, as determined with a bleeding score (BS). The secondary aim was to Correspondence: Anna Olsson, MD, Department of Haematology and Coagulation Disorders, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden. Tel: +46 31 3427342; fax: +46 31 820269; e-mail: [email protected] Accepted after revision 18 October 2014 © 2014 John Wiley & Sons Ltd
explore the correlation between TGA results and FVIII:C. Genetically identified adult carriers of severe and moderate haemophilia A were eligible to participate in the study [2]. Carriers registered at the three haemophilia centres in Sweden received a written invitation to participate. Written informed consent was obtained from all study participants. The study was approved by the Regional Ethical Review Board at the University of Gothenburg (450-09). A condensed version of the bleeding assessment tool (BAT) developed by the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 VWD study group was used to evaluate bleeding tendency among the carriers [6]. Each participant was interviewed by the same investigator and the structured questionnaires were summarized in a total BS, as reported previously [2]. A total BS < 4 is considered to indicate normal bleeding tendency [6]. Blood samples were collected by venipuncture using a 21-gauge needle into vacuum tubes (Vacutainer, Becton Dickinson, Plymouth, UK) containing 0.129 mol L 1 trisodium citrate without corn trypsin inhibitor (CTI). The blood was centrifuged at 2000 9 g for 20 min at room temperature within 30 min of collection. The citrated plasma was aliquoted and stored at 70°C. Factor VIII:C was measured with a two-stage amidolytic assay using the Coamatic FVIII kit (Chromogenix, Lexington, MA, USA). TGA was performed by the calibrated automated thrombin generation method (CAT) (Thrombinoscope BV, Maastricht, the Netherlands) according to the manufacturer’s instructions [7]. The TGA-related materials, i.e. calibrator (cat# TS20.00), PPP-low reagent [tissue factor (TF) at a final concentration of 1 pM and 4 lM phospholipids; cat# TS31.00], PPP reagent (TF at a final concentration of 5 pM and 4 lM phospholipids; cat#TS30.00) and FluCa solution (fluorogenic substrate and buffer; cat#TS50.00) were supplied by Thrombinoscope BV. Fluorescens was read in a Fluoroscan Ascent reader (Thermo Lab systems, Helsinki, 1
2
LETTER TO THE EDITOR
Finland) equipped with a 390/460 filter. The results were analysed using Thrombinoscope software version 5.0.0.742 (Thrombinoscope BV) and delivered as a thrombogram presenting the lag phase (min), time to peak (min), peak thrombin (nM) and endogenous thrombin potential (ETP, nM/min). The inter-assay coefficients of variation (CV%) across the TGA was measured using pooled normal plasma (CRYOcheck, Precision BioLogic, Dartmouth, Canada) resulting in CV% 8–14% for the lag phase, time to peak, peak and ETP when triggered with 1 pM TF and 10–19% when triggered with 5 pM TF. Statistical analyses were performed using SPSS, version 19.0 (SPSS, Chicago IL, USA). The Mann–Whitney U-test was used to detect differences in results between groups of carriers. A P-value of less than 0.05 was considered significant. One hundred and six carriers of severe and moderate haemophilia A were included in the study. Thirtyfive (33%) of the carriers had elevated BS (≥4). The characteristics of the carriers, grouped according to BS Table 1. Characteristics of participating haemophilia A carriers. BS < 4 n = 71 Age (years; median, range) Bleeding score (median, range) FVIII:C (kIU L 1)* (R: 0.50–2.00) VWF:RCo (kIU L 1)* (R: 0.40–1.20) Fibrinogen (g L 1)* (R: 2.0–4.5) APTT (s)* (R: 30–42) PT (INR)* (R:
DOI: 10.1111/hae.12585
LETTER TO THE EDITOR
Bleeding phenotype in carriers of haemophilia A does not correlate with thrombin generation € ¶ and F . B A G H A E I* A . O L S S O N , * M . H E L L G R E N , † ‡ E . B E R N T O R P , § M . H O L M S T R OM *Department of Haematology and Coagulation Disorders, Sahlgrenska University Hospital, Gothenburg; †Department of Antenatal Care N€ arh€ alsan Primary Care, V€ astra G€ otaland; ‡Department of Obstetrics and Gynaecology Institute of Clinical Science, Sahlgrenska Academy, University of Gothenburg, Gothenburg; §Centre for Thrombosis and Haemostasis Sk ane University Hospital, Lund University, Malm€ o; and ¶Coagulation Unit Haematology Centre, Karolinska University Hospital, Stockholm, Sweden
Haemophilia A is an X-linked disorder. Females who inherit the mutation are known as haemophilia A carriers. Clotting factor level in carriers are typically about 50% of levels in the normal population, but may vary considerably due to random X inactivation. Studies have reported increased bleeding tendency among carriers, especially in situations that challenge haemostasis, such as surgery [1]. In our previous study [2], we found an increased bleeding tendency in a cohort of carriers of moderate and severe haemophilia. Bleeding tendency was weakly correlated with clotting factor levels in haemophilia A carriers, but there was nonetheless a group of carriers with increased bleeding tendency despite normal levels. The reverse condition, a normal bleeding tendency combined with lowered factor levels, also existed in a subgroup of the carriers. This suggests that clotting factor levels alone may not be sufficient to reflect or predict bleeding tendency among carriers. Thrombin is a key enzyme in the coagulation process and thrombin generation assays (TGA) are used as a measure of global haemostasis. The thrombin generation profile of haemophilia A has been characterized [3,4] and TGA has illustrated the clinical heterogeneity among individuals with haemophilia with the same clotting factor levels [5]. Since clotting factor levels did not adequately predict bleeding risk in our cohort of carriers, we wanted to investigate whether measuring overall haemostasis with TGA might be more appropriate. The primary aim of this study was to compare TGA results in carriers of moderate and severe haemophilia A with increased bleeding tendency with those in carriers without increased bleeding tendency, as determined with a bleeding score (BS). The secondary aim was to Correspondence: Anna Olsson, MD, Department of Haematology and Coagulation Disorders, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden. Tel: +46 31 3427342; fax: +46 31 820269; e-mail: [email protected] Accepted after revision 18 October 2014 © 2014 John Wiley & Sons Ltd
explore the correlation between TGA results and FVIII:C. Genetically identified adult carriers of severe and moderate haemophilia A were eligible to participate in the study [2]. Carriers registered at the three haemophilia centres in Sweden received a written invitation to participate. Written informed consent was obtained from all study participants. The study was approved by the Regional Ethical Review Board at the University of Gothenburg (450-09). A condensed version of the bleeding assessment tool (BAT) developed by the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 VWD study group was used to evaluate bleeding tendency among the carriers [6]. Each participant was interviewed by the same investigator and the structured questionnaires were summarized in a total BS, as reported previously [2]. A total BS < 4 is considered to indicate normal bleeding tendency [6]. Blood samples were collected by venipuncture using a 21-gauge needle into vacuum tubes (Vacutainer, Becton Dickinson, Plymouth, UK) containing 0.129 mol L 1 trisodium citrate without corn trypsin inhibitor (CTI). The blood was centrifuged at 2000 9 g for 20 min at room temperature within 30 min of collection. The citrated plasma was aliquoted and stored at 70°C. Factor VIII:C was measured with a two-stage amidolytic assay using the Coamatic FVIII kit (Chromogenix, Lexington, MA, USA). TGA was performed by the calibrated automated thrombin generation method (CAT) (Thrombinoscope BV, Maastricht, the Netherlands) according to the manufacturer’s instructions [7]. The TGA-related materials, i.e. calibrator (cat# TS20.00), PPP-low reagent [tissue factor (TF) at a final concentration of 1 pM and 4 lM phospholipids; cat# TS31.00], PPP reagent (TF at a final concentration of 5 pM and 4 lM phospholipids; cat#TS30.00) and FluCa solution (fluorogenic substrate and buffer; cat#TS50.00) were supplied by Thrombinoscope BV. Fluorescens was read in a Fluoroscan Ascent reader (Thermo Lab systems, Helsinki, 1
2
LETTER TO THE EDITOR
Finland) equipped with a 390/460 filter. The results were analysed using Thrombinoscope software version 5.0.0.742 (Thrombinoscope BV) and delivered as a thrombogram presenting the lag phase (min), time to peak (min), peak thrombin (nM) and endogenous thrombin potential (ETP, nM/min). The inter-assay coefficients of variation (CV%) across the TGA was measured using pooled normal plasma (CRYOcheck, Precision BioLogic, Dartmouth, Canada) resulting in CV% 8–14% for the lag phase, time to peak, peak and ETP when triggered with 1 pM TF and 10–19% when triggered with 5 pM TF. Statistical analyses were performed using SPSS, version 19.0 (SPSS, Chicago IL, USA). The Mann–Whitney U-test was used to detect differences in results between groups of carriers. A P-value of less than 0.05 was considered significant. One hundred and six carriers of severe and moderate haemophilia A were included in the study. Thirtyfive (33%) of the carriers had elevated BS (≥4). The characteristics of the carriers, grouped according to BS Table 1. Characteristics of participating haemophilia A carriers. BS < 4 n = 71 Age (years; median, range) Bleeding score (median, range) FVIII:C (kIU L 1)* (R: 0.50–2.00) VWF:RCo (kIU L 1)* (R: 0.40–1.20) Fibrinogen (g L 1)* (R: 2.0–4.5) APTT (s)* (R: 30–42) PT (INR)* (R:
