A patient with type 2A von Willebrand disease and a long history of gastrointestinal (GI) bleeding is presented, in whom no abnormality was found on sequencing the von Willebrand factor gene at the DNA level. Subsequent RNA analysis revealed him to be heterozygous for a T-C substitution at nucleotide 4883, a mutation previously described and associated with type 2A von Willebrand disease. This illustrates the value of a dual DNA/RNA approach to genetic investigations of highly polymorphic genes. GI bleeding from angiodysplasia is a feature of von Willebrand disease, particularly type 2A. Proactive management with definitive diagnosis of angiodysplasia and ablative treatment where feasible is recommended to stop bleeding symptoms and minimize exposure to blood products.
A simple, rapid and cost-effective method for the analysis of three of the most widely screened genetic risk factors for thrombosis has been established. The protocol developed uses blood spots stored on filter paper (Guthrie spots) as well as DNA extracted from anticoagulated blood. The use of Guthrie spots taken at birth enables the retrospective study of patients who develop thrombotic complications without necessitating resampling. Following isolation of DNA, conventional fluorescence-labelled polymerase chain reaction (PCR) is performed using a thermostable DNA polymerase. Denatured, single-stranded PCR products are analysed on a semi-automated capillary-based genetic analyser, the data being stored electronically. This sensitive protocol obviates the need for endonuclease digestion and the associated gel running and documentation, and leads to a reduction in the recurrent costs of laboratory consumables.
Thrombotic thrombocytopenic purpura (TTP) is a rare disorder, with an annual incidence of 1–2/1 000 000. If untreated, mortality is 90% but with early plasma exchange (PEX), this can be reduced to 10–20%. The British Committee for Standards in Haematology (BCSH) guidelines recommend PEX within 4–8 h of the diagnosis of TTP being suspected (Amorosi & Ultmann, 1966; Rock et al, 1991; Terrell et al, 2005; Scully et al, 2012). Early treatment of TTP is crucial as around half of all deaths will occur within 24 h of presentation without treatment (Scully et al, 2008). Given that TTP can be difficult to distinguish clinically from other thrombotic microangiopathies (TMA), empirical PEX is mandated where TTP is a possibility in a patient with a TMA. Therefore, several days of PEX can therefore be unnecessarily given to patients who are eventually diagnosed not to have TTP. The laboratory diagnosis of TTP is confirmed by a Disintegrin And Metalloproteinase with a Thrombospondin type 1 motif, member 13 (ADAMTS13) activity level of <5%, but, until recently, this assay has only been available in specialised centres, with slow turnaround times as many centres only perform weekly runs. A rapid ADAMTS13 activity assay would be invaluable for diagnostic and treatment decisions. In 2015, our institution introduced a rapid automated ADAMTS13 activity assay that is available 24 h per day. This study investigated the effect of this rapid ADAMTS13 assay on patient management, outcomes, safety and potential resource savings. A retrospective audit, registered on the hospital quality system, of requests for rapid ADAMTS13 activity assay (turn around 4–6 h) between 1 September 2015 and 20 June 2017, was performed. Patients were identified from the laboratory database of sample requests. Clinical parameters collected included: treatment, platelet count, ADAMTS13 activity, intensive care unit (ICU) admission, final diagnosis and whether the patient was alive. ADAMTS13 activity was assessed by Technozym ADAMTS13 chromogenic enzyme-linked immunosorbent assay (ELISA; Pathway Diagnostics Ltd, Dorking, Surrey, UK) on a Dynex DS2 analyser (Werfen UK, Warrington, Cheshire, UK). The inter- and intra-assay coefficient of variance (CV) was 3·9% (n = 5) and 2·2% (n = 10), respectively. Where ADAMTS13 activity was <5%, the presence of anti-ADAMTS13 IgG was assessed by Technozym ADAMTS13 chromogenic ELISA (Pathway Diagnostics Ltd) on a Dynex DS2 analyser (Werfen UK,) The inter- and intra-assay CVs were 3·1% (n = 4) and 6·0% (n = 10), respectively. Patients with congenital TTP (i.e. with ADAMTS13 activity <5% without detectable inhibitor) underwent confirmatory genetic studies by ADAMTS13 gene sequencing. There were 22 requests for urgent, rapid ADAMTS13 activity results with a turnaround time of 4·5–6·0 h (median 5 h). 1 non-TTP case from another hospital was excluded due to lack of clinical information. Of the 21 cases studied, seven (33%) had TTP, defined as ADAMTS13 activity <5% and were tested for anti-ADAMTS13 IgG (Table 1). The remaining 14 patients had a range of TMAs (Table 2). The median ADAMTS13 activity in the 14 non-TTP patients was 61·5% (range 36–104%; normal range 66–107%); four patients had low level ADAMTS13 activity (10–40%), currently thought to represent increased consumption (Nguyen et al, 2007). Median platelet count in non-TTP patients was 34 × 109/l (range 5–142 × 109/l; normal range 150–400 × 109/l). Seven of the 14 (50%) non-TTP patients were not in the ICU when the sample for ADAMTS13 activity was sent and were not admitted to ICU whilst the result was awaited as, at our institution, all patients with suspected TTP requiring PEX are admitted to ICU. Given that an ICU bed was estimated to cost £1932 per day compared to £413 for a ward bed (NHS Wales, 2013), preventing ICU admissions has potential cost savings, as well as saving on staff time and opportunity costs in preventing elective surgery cancellations where an ICU bed is unnecessarily occupied. In nine of the 14 (65%) non-TTP cases, neither PEX or plasma infusions were initiated as the diagnosis was equivocal and clinicians felt the risks of waiting 4-6 h for the ADAMTS13 results were acceptable. In the other cases (n = 5) a central line was inserted, but PEX was stopped after one exchange in two patients, after two exchanges in one patient, and one patient had an infusion of solvent detergent treated fresh frozen plasma (SD-FFP) prior to the ADAMTS13 result being available. A typical PEX uses 1–1·5 plasma volume exchange. BCSH guidance recommends the use of SD-FFP for PEX (O'Shaughnessy et al, 2004). A unit of Octaplas (Octapharma, Charlotte, NC, USA), the SD-FFP used at our institution costs £65.00, and a 1·5-volume PEX in a 70-kg man would typically use a minimum of 14 units of Octaplas, so a cost saving of at least £910.00 in SD-FFP would be made for every PEX prevented. In this study, an estimated £8190 in SD-FFP was saved by preventing one-off exchanges. Equipment costs (e.g. central line and apharesis equipment) would also be saved. Complications of plasma infusion include transfusion-related acute lung injury, transfusion-associated cardiac overload, pathogen transmission, severe allergic reactions and febrile reactions. Pulmonary complications of plasma infusion were the leading cause of death in the United Kingdom Serious Hazards Of Transfusion for 2010-16, accounting for 53% of deaths related to transfusion (Bolton-Maggs et al, 2017). Other complications of PEX are intravascular fluid shift between compartments, central venous access, citrate toxicity and hypotension. Avoiding such complications in already critically ill patients is highly desirable. Delayed PEX causes preventable deaths in TTP and, in this study, genuine TTP patients with acute disease had PEX started before the ADAMTS13 activity result was known, showing it does not cause treatment delay but allows early diagnostic refinement (Pereira et al, 1995). However, in patients whose eventual diagnosis was not TTP, the rapid ADAMTS13 result prevented inappropriate administration of plasma and unnecessary ICU admission. In conclusion, rapid ADAMTS13 activity assay is useful in excluding TTP in patients with TMA where there is diagnostic uncertainty. This is helpful where clinicians have little clinical exposure to TTP and lack confidence in diagnosis. In patients whose eventual diagnosis was not TTP, the rapid ADAMTS13 result prevented inappropriate administration of plasma and unnecessary ICU admission. It seems a rapid ADAMTS13 assay is a step forward in diagnosis of TTP and has potential cost savings. Drs Thomas, McDonald and Hunt jointly wrote the manuscript and Drs Cutler and Moore set up the assay and agreed the final manuscript.
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