Gillian Evans

Thrombotic thrombocytopenic purpura and pregnancy: presentation, management, and subsequent pregnancy outcomes

Pregnancy can precipitate thrombotic thrombocytopenic purpura (TTP). We present a prospective study of TTP cases from the United Kingdom Thrombotic Thrombocytopenic Purpura (UK TTP) Registry with clinical and laboratory data from the largest cohort of pregnancy-associated TTP and describe management through pregnancy, averting fetal loss and maternal complications. Thirty-five women presented with a first TTP episode during pregnancy: 23/47 with their first congenital TTP (cTTP) episode and 12/47 with acute acquired TTP in pregnancy. TTP presented primarily in the third trimester/postpartum, but fetal loss was highest in the second trimester. Fetal loss occurred in 16/38 pregnancies before cTTP was diagnosed, but in none of the 15 subsequent managed pregnancies. Seventeen of 23 congenital cases had a missense mutation, C3178T, within exon 24 (R1060W). There were 8 novel mutations. In acquired TTP presentations, fetal loss occurred in 5/18 pregnancies and 2 terminations because of disease. We also present data on 12 women with a history of nonpregnancy-associated TTP: 18 subsequent pregnancies have been successfully managed, guided by ADAMTS13 levels. cTTP presents more frequently than acquired TTP during pregnancy and must be differentiated by ADAMTS13 analysis. Careful diagnosis, monitoring, and treatment in congenital and acquired TTP have assisted in excellent pregnancy outcomes.

Thrombin generation: a comparison of assays using platelet-poor and -rich plasma and whole blood samples from healthy controls and patients with a history of venous thromboembolism

We have developed a whole blood thrombin generation (TG) assay whereby TG is initiated with a low‐tissue factor concentration and monitored using a fluorogenic thrombin substrate. Significantly higher values were found in blood samples from 50 patients with a history of venous thromboembolism (VTE) compared with 31 healthy controls (HC), for peak height (P = 0·0034) and endogenous thrombin potential (ETP) (P = 0·0027). Results from 31 VTE patients and the 31 controls in the absence of corn trypsin inhibitor (CTI) showed significantly higher values in the VTE group for peak height (P = 0·0013) and ETP (P = 0·002). In the presence of CTI, significantly higher values were only seen in ETP (P = 0·024). No significant increases in TG were found using platelet poor (PPP) or ‐rich (PRP) plasma with or without CTI. The whole blood TG assay in the absence or presence of CTI showed a higher proportion (25/50 and 12/31, respectively) of raised peak height and/or ETP values than plasma assays (PPP 9/50 and 5/31 respectively and PRP 13/50 and 6/31, respectively). Our results show the whole blood TG assay is more sensitive for determining the increases in TG in patients with a history of VTE than PPP and PRP TG assays.

Tracking and characterisation of transfused platelets by two colour, whole blood flow cytometry

We describe a flow cytometric technique to study transfused platelets in patients. By selecting donor/recipient pairs discrepant for HLA‐A2, transfused platelets were identified using anti‐HLA‐A2 with a detection limit of <5%, and accuracy within 4·3% of predicted (r2 > 0·96, P < 0·0001). In two of three episodes, transfused platelets were present in greater numbers than predicted from increment counts. Platelets with bound fibrinogen fell from 20·9 ± 23·6% of donor platelets pretransfusion to 1·7 ± 0·3% 1 h post‐transfusion, whereas P‐selectin‐positive platelets fell gradually, from 24·1 ± 6·7 to 7·3 ± 3·3% at 6 h. This method avoids radio or chemical labelling, and could be used to assess novel platelet preparations post‐transfusion.

Autoimmune neutropenia following therapy for chronic lymphocytic leukaemia: a report of three cases.

Rubnitz, J.E., Razzouk, B., Relling, M.V., Evans, W.E., Boyett, J.M. & Pui, C.H. (2000) Traumatic lumbar puncture at diagnosis adversely affects outcome in childhood acute lymphoblastic leukaemia. Blood, 96, 3381–3384. Howard, S.C., Gajjar, A., Ribeiro, G.K., Rivera, G.K., Rubnitz, J.E., Sandlund, J.T., Harrison, P.L., de Armendi, A., Dahl, G.V. & Pui, C.H.. (2000) Safety of lumbar puncture for children with acute lymphoblastic leukaemia and thrombocytopenia. Journal of American Medical Association, 284, 2222–2224. Howard, S.C., Gajjar, A.J., Cheng, C., Kritchevsky, S.B., Somes, G.W., Harrison, P.L., Ribeiro, R.C., Rivera, G.K., Rubnitz, J.E., Sandlund, J.T., de Armendi, A.J., Razzouk, B.I. & Pui, C.H. (2002) Risk factors for traumatic and bloody lumbar puncture with acute lymphoblastic leukaemia. Journal of American Medical Association, 288, 2001–2007.

Endoscopy and the Risk of Venous Thromboembolism: A Case-Control Study

Background and Study Aims To assess whether there was an association between endoscopy and the risk of venous thromboembolism (VTE). Patients and Methods Retrospective case – control study of patients diagnosed with VTE over a 3-year period. Each was age- and sex-matched to one of three controls who attended an outpatient appointment on the same date as that of the diagnosis of VTE in the patients. Patients who had undergone endoscopy within 90 days of VTE were included. On a second analysis, patients who were hospitalized and those with inflammatory bowel disease or malignancy were excluded. The difference in occurrence of endoscopy between cases and controls was examined using the McNemar test. The risk of VTE occurring following endoscopy was quantified by means of odds ratios. Results Forty-five of 436 patients (10.3 %) had undergone an endoscopy in the VTE group compared with 14 /436 controls (3.2 %; P < 0.001). The odds ratio for developing a VTE after an endoscopic procedure was 3.58 (95 % CI 1.86 – 7.46) for patients relative to controls. When the 10 hospitalized patients and respective controls were excluded, the odds of VTE remained nearly 3 times as large for patients undergoing endoscopy as for controls (2.92 [95 % CI 1.51, 5.62]; P = 0.001). When patients with inflammatory bowel disease or malignancy were also excluded, no difference was found between patients undergoing endoscopy and controls (1.92 [0.95, 3.85]; P = 0.07). Ten percent of patients with VTE underwent endoscopy in the 3 months before the diagnosis compared with 3 % of controls (P < 0.001). No significant difference was found between the type of endoscopy performed and VTE risk. Conclusions When those with known risk factors for VTE were excluded, no significant increased risk of VTE was found.

A United Kingdom Immune Thrombocytopenia (ITP) Forum review of practice: thrombopoietin receptor agonists

Tothova, E., Keil, F., Autzinger, E.M., Thaler, J., Gisslinger, H., Lang, A., Egyed, M., Womastek, I. & Zojer, N. (2010) Light chaininduced acute renal failure can be reversed by bortezomib-doxorubicin-dexamethasone in multiple myeloma: results of a phase II study. Journal of Clinical Oncology, 28, 4635– 4641. Morgan, G.J., Davies, F.E., Gregory, W.M., Bell, S.E., Szubert, A.J., Cook, G., Drayson, M.T., Owen, R.G., Ross, F.M., Jackson, G.H. & Child, J.A. (2013) Long-term follow-up of MRC Myeloma IX trial: survival outcomes with bisphosphonate and thalidomide treatment. Clinical Cancer Research, 19, 6030–6038. Palumbo, A., Cavallo, F., Gay, F., Di Raimondo, F., Ben Yehuda, D., Petrucci, M.T., Pezzatti, S., Caravita, T., Cerrato, C., Ribakovsky, E., Genuardi, M., Cafro, A., Marcatti, M., Catalano, L., Offidani, M., Carella, A.M., Zamagni, E., Patriarca, F., Musto, P., Evangelista, A., Ciccone, G., Omede, P., Crippa, C., Corradini, P., Nagler, A., Boccadoro, M. & Cavo, M. (2014) Autologous transplantation and maintenance therapy in multiple myeloma. New England Journal of Medicine, 371, 895–905.

A comprehensive targeted next-generation sequencing panel for genetic diagnosis of patients with suspected inherited thrombocytopenia

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet counts and often disproportionate bleeding with over 30 genes currently implicated. Previously the UK‐GAPP study using whole exome sequencing (WES) identified a pathogenic variant in 19 of 47 (40%) patients of which 71% had variants in genes known to cause IT.

Prostate cancer: beware of disseminated intravascular coagulation

Disseminated intravascular coagulation (DIC) is a pathological systemic condition resulting from aberrant activation of the coagulation system. It is characterised by the release and activation of procoagulants into the blood, with an associated consumption coagulopathy. Its association with solid and haematological malignancies is well described in literature. This case describes an elderly man, known to have prostate cancer, who following transurethral resection of the prostate developed DIC with haematuria, spontaneous ecchymoses and mucosal bleeding. Subsequent investigations revealed a prostate-specific antigen (PSA) >1000 µg/L, and staging CT showed multiple sclerotic metastatic lesions affecting the thoracic and lumbar vertebra, as well as infiltration into his left femur. Coagulation normalised with blood products and vitamin K within 1 week, and the patient responded to antiandrogen therapy with a reduction in pain and PSA on discharge.

Thrombin generation in whole blood – response to Al Dieri & Hemker

We read with interest the comments by Al Dieri and Hemker on our recent article on thrombin generation (TG) in plateletpoor plasma (PPP), platelet-rich plasma (PRP) and whole blood (WB) (Tappenden et al, 2007). Al Dieri and Hemker report that they have tried unsuccessfully to reproduce our method for determining TG in WB. They suggest that, because of this, we must have omitted one or more details of the experimental procedures in our paper. They base their argument on two major points. Their first point deals with the fluorescent signals obtained in WB. They state that they found these to be 30· weaker in WB than those in plasma and that the signal to noise ratio was so poor in WB that they were unable to obtain useful first derivatives. The results described in our paper follow over 2 years development work on the plasma-based and WB TG methods including comparisons of different microtitre plates (clear or black and shape), studies on a variety of fluorogenic peptide substrates, the determination of the actual amounts of tissue factor added in the assays and excitation and emission wavelength scans to find optimal assay conditions. These studies were performed using a Gemini XS fluorimeter (Molecular Devices, West Sussex, UK), which is equipped with a monochromator rather than a filter wheel and has the ability to mix the incubating material between readings. In our method the samples were read every 30 s and continually mixed during the incubation period between readings. A major influence on obtaining results in WB was the use of different excitation and emission wavelengths for WB compared to those used for PPP and PRP. Using the wavelengths specified the signal obtained for WB was two to three times weaker than that seen in plasma samples. We were able to obtain relative fluorescence units (RFUs) in excess of 3500 in WB. This produced a signal to noise ratio (at an RFU of 2600) some 3· poorer than that obtained in PPP. This did not however prevent the calculation of the first derivative. The second point in the letter by Hemker and Al Dieri deals with erratic TG curves obtained with WB and the conversion of the fluorescence data. While the curves obtained in the TG assay are more erratic in WB than those in PPP (Fig 1A and B) they still allow for conversion to the first derivative and thus the calculation of the thrombin concentration. In those samples where the fluorescence is higher than that of the calibrator, the effect may be an underestimation of the inner filter effect; this may reduce the final thrombin concentration calculated by the mathematical model. It is worth noting that in comments made by AlDieri and Hemker they fail to state the type of fluorimeter used in their experiments. We believe the reason for our success in measuring TG in WB is not only because of the optimized assay conditions and wavelengths chosen, but also to the type of fluorimeter used. We surmise that this is probably as a result of the fluorimeter using a monochromator rather than a filter wheel but there may be other properties of this particular instrument that have contributed to our success. We have no way of proving this conclusively as we only have access to this one instrument. There may also be other factors influencing our ability to measure TG in WB, as it is well known that the results of assays for TG can be influenced dramatically by quite minor changes in technique. The point made by Al Dieri and Hemker about clot formation and clot retraction is important. However, the type of plastic used in microtitre plates may differ considerably (A)