Sheffield Haemophilia and Thrombosis Center

Sheffield, United Kingdom

Sheffield Haemophilia and Thrombosis Center

Sheffield, United Kingdom

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Kitchen S.,Sheffield Haemophilia and Thrombosis Center | Kershaw G.,Royal Prince Alfred Hospital | Tiefenbacher S.,Laboratory Corporation of America
Haemophilia | Year: 2016

The recent development of modified recombinant factor VIII (FVIII) and factor IX (FIX) therapeutic products with extended half-lives will create challenges for the haemostasis laboratory in obtaining recovery estimates of these products in clinical samples using existing assays. The new long-acting therapeutic concentrates contain molecular modifications of Fc fusion, site-specific of polyethylene glycol or albumin fusion. The optimum methods for monitoring each new product will need to be assessed individually and laboratories should select an assay which gives similar results to the assay used to assign potency to the product in question. For some extended half-life FVIII and FIX products some one stage assays are entirely unsuitable for monitoring purposes. For most products and assay reagents studied so far, and reviewed in this manuscript, chromogenic FVIII or FIX assays can be safely used with conventional plasma standards. If one stage assays are used then they should be performed using carefully selected reagents/methods which have been shown to recover activity close to the labelled potency for the specific product being monitored. © 2016 John Wiley & Sons Ltd


Bowyer A.,Sheffield Haemophilia and Thrombosis Center | Bowyer A.,University of Sheffield | Kitchen S.,Sheffield Haemophilia and Thrombosis Center | Makris M.,Sheffield Haemophilia and Thrombosis Center | Makris M.,University of Sheffield
International Journal of Laboratory Hematology | Year: 2011

Introduction: The sensitivity of APTT reagents to deficiencies of factors VIII, IX, XI and XII varies because of their composition. The APTT is used as a screening test for these factors, and a deficiency should manifest with a prolongation to the APTT, which may trigger the need for specific factor assays to be performed. Methods: The suitability of APTT reagents to detect mild deficiencies can be assessed by the analysis of the APTT of plasma, which has an increasing concentration of the factor in question. The APTT responsiveness can be determined from the intersection of the curve and the upper limit of the APTT normal reference range for that APTT reagent. We assessed the APTT responsiveness (in U/dl) to factors VIII, IX and XI of four APTT reagents; Actin FS (Siemens), Synthasil (IL), STA-PTTA (Stago) and Dapttin (Technoclone). Results: Actin FS was the most sensitive reagent to mild reductions of factors VIII, IX and XI [Correction added on 26 October 2010, after first online publication: Synthasil was corrected to Actin FS]. STA-PTTA showed less sensitivity than Synthasil and Actin FS; Dapttin was insensitive to mild deficiencies of factors IX and XI and should not be used as a screening test. Conclusion: Both Synthasil and Actin FS are acceptable reagents to screen for reduced factors VIII, IX and XI, and the number of mildly reduced factors not diagnosed will be limited. © 2010 Blackwell Publishing Ltd.


Cooper P.C.,Sheffield Haemophilia and Thrombosis Center | Hill M.,University of Nottingham | Maclean R.M.,Sheffield Haemophilia and Thrombosis Center
International Journal of Laboratory Hematology | Year: 2012

This paper outlines the methods and approaches used for the laboratory detection and investigation of protein C (PC) deficiency. It does not make recommendations as to which patients should have thrombophilia testing performed; this should be done in line with local guidance. Interpretation of PC level is complicated because level varies with age, and many conditions can cause acquired deficiency. Protein C is most usually measured by chromogenic assay as a part of the thrombophilia screen. There exists, however, a very small group of individuals with significant PC deficiency, in whom the chromogenic PC assay is normal. The coagulometric assay of PC is more sensitive to these rare defects, but these assays may lack specificity. Genetic analysis allows definitive diagnosis and may be useful in confirming that deficiency is inherited and not acquired and is particularly valuable in families with severe PC deficiency. © 2012 Blackwell Publishing Ltd.


Bowyer A.E.,Sheffield Haemophilia and Thrombosis Center | Goodeve A.,Sheffield Childrens NHS Foundation Trust | Goodeve A.,University of Sheffield | Liesner R.,Great Ormond Street Hospital for Children | And 4 more authors.
British Journal of Haematology | Year: 2011

Haemophilia A is caused by a reduction in clotting factor VIII (FVIII). FVIII coagulant activity (FVIII:C) can be measured by three methods; the one-stage activated partial thromboplastin time-based clotting assay, the two-stage Xa generation-based clotting assay and the chromogenic Xa generation-based assay. The FVIII:C of most patients with haemophilia A are concordant regardless of the assay method employed. Up to a third of patients show assay discrepancy, usually with the two-stage and chromogenic assays being much lower than the one-stage assay. Very rarely, patients have been reported with lower one-stage compared to two-stage and chromogenic assays, but here we report that the mutation p.Tyr365Cys accounts for most of these patients and, at least in the UK, is not rare. We have identified this mutation in 23 different families. Affected male index individuals had a lower mean one-stage FVIII:C of 27iu/dl compared to two-stage FVIII:C mean of 77iu/dl. Affected individuals had minimal or absent bleeding symptoms and when these were present they were usually in patients with another co-inherited bleeding disorder. Affected individuals often had surgery without the need to correct the one-stage FVIII:C. © 2011 Blackwell Publishing Ltd.


Cooper P.C.,Sheffield Haemophilia and Thrombosis Center | Siddiq S.,University of Bristol | Morse C.,University of Bristol | Nightingale J.,University of Canterbury | Mumford A.D.,University of Bristol
International Journal of Laboratory Hematology | Year: 2011

Introduction: Protein C (PC) Asn2Ile is a rare pro-thrombotic variant in which loss of anticoagulant function in vivo is likely to result from defective PC binding to the endothelial PC receptor and phospholipid. Methods: To characterize the consequences of this functional defect on the diagnostic performance of commercial PC activity assays, we performed one antigen, three chromogenic and three coagulometric assays on plasma from a subject who was heterozygous for the PC Asn2Ile substitution. Results: As anticipated, the PC antigen (96.2IU/dl) and chromogenic PC activity assays (98.4IU/dl) were insensitive to PC Asn2Ile, which has intact enzymatic activity and is secreted normally. There was a marked reduction in apparent PC activity by STACLOT coagulometric assay (50.4IU/dl). However, there was only a small reduction in apparent PC activity by CRYOcheck Clot C assay (69.5IU/dl) and by HemosIL ProClot assay, the PC activity was within the laboratory reference range (91IU/dl). This discrepancy between coagulometric assays could not be explained by lupus anticoagulant, activated PC resistance or elevated plasma factor VIII activity. Conclusion: We demonstrate that coagulometric assays are not equally sensitive to clinically important functional defects of PC and that multiple assays may be required to identify all variants. © 2011 Blackwell Publishing Ltd.


Toth P.,Sheffield Haemophilia and Thrombosis Center | Van Veen J.J.,Sheffield Haemophilia and Thrombosis Center | Robinson K.,Sheffield Haemophilia and Thrombosis Center | Maclean R.M.,Sheffield Haemophilia and Thrombosis Center | And 4 more authors.
Blood Transfusion | Year: 2013

Background. Life threatening bleeding and emergency procedures in patients on vitamin K antagonists are indications for urgent reversal with prothrombin complex concentrate and vitamin K. Rapid reversal in these situations is emphasized in the literature and guidelines, but only very limited information is available on its real life use, especially on the timing of treatment in relation to presentation. Materials and methods. We retrospectively audited emergency warfarin reversal in 131 consecutive patients. We studied the indication, use of vitamin K, time between presentation and administration of vitamin K and PCC, effectiveness in INR reduction and clinical outcome. Results. The median PCC dose was 26.8 IU/kg. The median INR was reduced from 3.1 to 1.2. Vitamin K (5 mg) was given in 91.6% of evaluable patients. We found significant delays in administration of PCC and vitamin K. The median time between presentation and administration of vitamin K/PCC was 3.6 and 5.2 hours respectively. The times in intracranial haemorrhage were 2.7 and 3.0 hours and in emergency procedures 17.4 and 15.9 hours respectively. Mortality related to bleeding was 7.6% overall but in patients with intracranial haemorrhage 22.8%. The thrombotic rate within 7 days of reversal was 1.5%. Discussion. The local protocol for reversal with PCC and vitamin K was adhered to well but the delay in pre-procedural patients, suggests that intravenous vitamin K alone may be sufficient in many cases and PCC administration can be avoided by better planning. Intracranial haemorrhage in warfarinised patients carries a high mortality. Treatment delays should be avoided by making PCC stocks available within emergency departments, simple dosing structures independent of INR and administering PCC without waiting for INR and CT scan results in those with strong suspicion of intracranial haemorrhage and clear trauma. Future reports and studies should always include the time from presentation to PCC treatment. © SIMTI Servizi Srl.


Cooper P.C.,Sheffield Haemophilia and Thrombosis Center | Goodeve A.C.,University of Sheffield | Goodeve A.C.,Sheffield Childrens NHS Foundation Trust | Beauchamp N.J.,Sheffield Childrens NHS Foundation Trust
Seminars in Thrombosis and Hemostasis | Year: 2012

As the understanding of the genetic basis of the inherited thrombophilias has increased over recent years, their routine diagnostic genetic analysis has also matured. This review considers methods used to test for the factor V (F5) Leiden mutation and prothrombin 20210A (F2 c. *97G>A) allele, and analysis of the SERPINC1, PROC, and PROS1 genes in cases of antithrombin, protein C (PC), and protein S (PS) deficiency, respectively. Issues relating to quality are explored, highlighting where analytical and sample handling errors may occur. Detection of the factor V Leiden mutation and the prothrombin c.*97G>A allele are best performed using real-time polymerase chain reaction analysis as this relatively simple technique allows their discrimination from rare variants of neighboring nucleotides; not possible using the more time-consuming restriction digestion assays. With the advent of low-cost and high-throughput sequence analysis, direct sequencing has become the first-line method to provide a definitive diagnosis of inherited, rather than acquired, deficiencies. Large cohort studies have shown that antithrombin and PC mutations are identified in between 61 and 87% of patients, whereas the detection rate in PS deficiency is substantially lower in around 40% of patients. Large gene deletions make up between 7 and 10% of PS and antithrombin mutations and only 1% of PC mutations, but it is suggested that dosage analysis techniques such as multiplex ligation-dependent probe amplification should be used for all three genes as part of routine analysis to ensure mutations are not missed. Best practice guidelines are available from EuroGentest covering a wide variety of the issues raised in this review and all laboratories should participate in appropriate external quality assurance schemes to ensure they continue to offer high quality service. © 2012 by Thieme Medical Publishers, Inc.


Bowyer A.E.,Sheffield Haemophilia and Thrombosis Center | Van Veen J.J.,Sheffield Haemophilia and Thrombosis Center | Goodeve A.C.,University of Sheffield | Goodeve A.C.,Sheffield Childrens NHS Foundation Trust | And 3 more authors.
Haematologica | Year: 2013

The activity of the factor VIII coagulation protein can be measured by three methods: a one or two-stage clotting assay and a chromogenic assay. The factor VIII activity of most individuals with mild hemophilia A is the same regardless of which method is employed. However, approximately 30% of patients show marked discrepancies in factor VIII activity measured with the different methods. The objective of this study was to investigate the incidence of assay discrepancy in our center, assess the impact of alternative reagents on factor VIII activity assays and determine the usefulness of global assays of hemostasis in mild hemophilia A. Factor VIII activity was measured in 84 individuals with mild hemophilia A using different reagents. Assay discrepancy was defined as a two-fold or greater difference between the results of the one-stage and two-stage clotting assays. Rotational thromboelastometry and calibrated automated thrombography were performed. Assay discrepancy was observed in 31% of individuals; 12% with lower activity in the two-stage assay and 19% with lower activity in the one-stage assay. The phenotype could not always be predicted from the individual's genotype. Chromogenic assays were shown to be a suitable alternative to the two-stage clotting assay. Thromboelastometry was found to have poor sensitivity in hemophilia. Calibrated automated thrombography supported the results obtained by the two-stage and chromogenic assays. The current international guidelines do not define the type of assay to be used in the diagnosis of mild hemophilia A and some patients could be misclassified as normal. In our study, 4% of patients would not have been diagnosed on the basis of the one-stage factor VIII assay. Laboratories should use both one stage and chromogenic (or two-stage) assays in the diagnosis of patients with possible hemophilia A. © 2013 Ferrata Storti Foundation.


Millar C.M.,Imperial College London | Makris M.,Sheffield Haemophilia and Thrombosis Center
British Journal of Haematology | Year: 2012

The identification of variant Creutzfeldt-Jakob disease (vCJD) in the UK in 1996 led to significant concerns about the possibility of secondary transmission, however the prevalence of subclinical vCJD and risks of vCJD transmission by plasma are not known. In the UK, public health precautions have been implemented in all recipients of coagulation factor concentrates manufactured from UK plasma pools between 1980 and 2001. The recent demonstration of abnormal prion protein in a spleen sample at autopsy of a UK haemophilic patient who received coagulation factor concentrates to which a donor incubating vCJD had contributed most likely represents the first case of vCJD transmission by coagulation factor concentrates. We review the uncertainties that surround risk of vCJD transmission by coagulation factor concentrates, the challenges in dealing with undefined risks, the rationale behind current policies and the implementation of vCJD surveillance and risk management measures in bleeding disorder patients in the UK. © 2012 Blackwell Publishing Ltd.


van Veen J.J.,Sheffield Haemophilia and Thrombosis Center | Maclean R.M.,Sheffield Haemophilia and Thrombosis Center | Hampton K.K.,Sheffield Haemophilia and Thrombosis Center | Hamer A.,Northern General Hospital | And 2 more authors.
Haemophilia | Year: 2014

Major surgery in persons with haemophilia A and inhibitors is increasingly being performed. Both recombinant activated factor VII (rFVIIa) and activated prothrombin complex concentrate (APCC) are used to cover surgery but it remains unclear what the optimal dosing schedules are. We describe the use of a hybrid regimen in four inhibitor patients undergoing eight major surgical procedures using rFVIIa in the initial 2-6 postoperative days followed by FEIBA® for the remaining period. All patients were also treated with tranexamic acid while receiving rFVIIa. We performed six major orthopaedic procedures, one emergency orchidectomy and one open appendectomy. The dosing schedules were at the higher end of those described in the literature but within the recommendations of the summary of product characteristics. Despite this, we encountered non-surgical bleeding in four of eight episodes. Three of these occurred in one individual suggesting a patient factor. The overall outcome was good for all episodes. The hybrid regimen combines flexibility of dose and dosing frequency of rFVIIa in the immediate postoperative setting with the advantage of a reduced dosing frequency with FEIBA® in the subsequent days. This study also emphasizes that surgical procedures in this patient group remain a challenge. © 2014 John Wiley & Sons Ltd.

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