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Keywords:

  • von Willebrand factor;
  • ADAMTS-13;
  • thrombotic thrombocytopenic purpura

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

The congenital or acquired deficiency of the von Willebrand factor (VWF) cleaving protease, ADAMTS-13 has been specifically associated with a diagnosis of thrombotic thrombocytopenic purpura (TTP), a microangiopathy characterized by the formation of occlusive platelet thrombi. The mechanisms of TTP were investigated in 100 patients diagnosed on the basis of the presence of at least three of the following: thrombocytopenia, haemolytic anaemia, elevated serum levels of lactate dehydrogenase and neurological symptoms. Plasma levels of ADAMTS-13 were severely reduced (<10% of normal) in 48%, moderately reduced (between 10% and 46%) in 24% and normal (>46%) in 28%. A neutralizing antibody was the cause of the deficiency in 38% of the cases, with a higher prevalence of this mechanism (87%) in the 48 patients with severely reduced ADAMTS-13. Double heterozygosity for a 29 base pair (bp) deletion and a nucleotide insertion and homozygosity for a 6 bp deletion in the ADAMTS13 gene were identified only in two patients born from consanguineous marriages. In conclusion, this study indicated that ADAMTS-13 was normal in nearly one-third of patients with TTP and that ADAMTS-13 deficiency was not associated with the presence of neutralizing antibodies in more than half of the patients.

Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) characterized by microvascular platelet aggregation, resulting in thrombocytopenia and a Coombs-negative haemolytic anaemia with fragmented erythrocytes, often accompanied by fluctuating organ dysfunction (particularly in the brain and kidney) (Moake, 2002). In TTP, the key mechanistic role of von Willebrand factor (VWF) – a multimeric adhesive protein contained in vascular endothelial cells, platelets and plasma – was first surmised by Moake et al (1982), who found that the plasmas of some patients with the chronic relapsing form of the disease contained ultralarge VWF multimers. These forms of VWF, absent in normal plasma, are secreted from activated endothelial cells and promote platelet-dependent microvascular thrombosis (Moake et al, 1986). A deficiency of a VWF-cleaving protease was postulated by Moake (2004) as the cause of the presence of ultralarge hyperactive VWF, but it was Furlan et al (1996) and Tsai (1996) partially purified, from human plasma, a metal-ion dependent protease that, in conditions of high shear stress, regulates the multimeric size of VWF by cleaving a specific peptide bond in the central A2 domain of VWF. The protease was identified as a new member of the ADAMTS family of metalloproteases (a disintegrin and metalloprotease with thrombospondins 1 repeats) and designated ADAMTS-13 (Fujikawa et al, 2001; Zheng et al, 2001).

The deficiency of ADAMTS-13 in TTP is congenital or acquired. The very rare congenital deficiency is an autosomal recessive condition because of compound heterozygous or homozygous mutations in the ADAMTS13 gene (Levy et al, 2001; Kokame et al, 2002; Schneppenheim et al, 2003; Kokame & Miyata, 2004; Veyradier et al, 2004). The more frequent acquired deficiency, caused by autoantibodies that neutralize ADAMTS-13 activity (Furlan et al, 1998; Tsai & Lian, 1998; Tsai et al, 2001) or bind to the protein causing its accelerated clearance from plasma (Scheiflinger et al, 2003), may be idiopathic or secondary to autoimmune diseases, pregnancy, metastatic cancer, drug intake, infections, sepsis and transplantation (particularly of allogeneic bone marrow and solid organs) (Moake, 2002).

The original studies of Furlan et al (1998) and Tsai and Lian (1998) claimed that very low or undetectable plasma levels of ADAMTS-13 specifically distinguish TTP from other TMAs, but the diagnostic specificity of ADAMTS-13 deficiency in TTP was subsequently questioned (Mannucci et al, 2001; Veyradier et al, 2001; Bianchi et al, 2002; Remuzzi et al, 2002; Vesely et al, 2003). With this background, we have performed a phenotypic and genotypic study in a large series of patients with TTP. The main goal of this study was to investigate the relationship between a clinical diagnosis of TTP, ADAMTS-13 activity, ADAMTS-13 neutralizing antibodies in plasma and ADAMTS13 gene defects.

Patients

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

Between May 2000 and April 2004 our tertiary referral Centre received requests from other clinical units – mainly from Italy and also from other countries (Germany, Greece, Iran, Slovenia, Turkey) – to assay protease and analyse DNA in a search for ADAMTS13 gene mutations. Each unit was required to provide citrated plasma and leucocyte samples for ADAMTS-13 assays and DNA analysis, with a completed questionnaire compiled to confirm or exclude a clinical diagnosis of TTP. We also encouraged clinicians to supply plasma collected in EDTA to enable analysis of the multimeric structure of VWF. Ethical approval of the study was granted by our Institutional Review Board.

A selection of the 140 samples that were initially received enabled the exclusion of a clinical diagnosis of TTP in some cases, in others it was established that samples were obtained after plasma therapy was started or inadequate clinical and laboratory data and/or samples were provided. Hence, the study was eventually limited to 100 untreated patients in whom a clinical diagnosis of acute TTP was confirmed on the basis of at least three of the following: thrombocytopenia, microangiopathic haemolytic anaemia, high serum lactate dehydrogenase (LDH) levels and neurological signs compatible with focal ischaemia. Before the study we decided to assay ADAMTS-13 activity in all patients with a confirmed clinical diagnosis of acute TTP and to look for anti-ADAMTS-13 neutralizing antibodies only in those who had plasma levels of ADAMTS-13 activity below the lower limit of the laboratory range (<46%). It was also arbitrarily decided to look for candidate gene mutations only in patients who had very low ADAMTS-13 levels (<20%), not associated with detectable anti-ADAMTS-13. Fig 1 summarizes the flow chart of the study.

image

Figure 1. Chart of the study protocol.

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Sample collection

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

For ADAMTS-13 activity and ADAMTS-13 inhibitor, blood was anticoagulated with sodium citrate, and with EDTA for multimeric analysis. As several centres did not supply plasma collected in EDTA, multimeric analysis was possible in 40 patients only. Platelet poor plasma was prepared by centrifugation, kept frozen at −80°C together with the buffy coat for DNA analysis and then sent in dry ice to the coordinating centre.

ADAMTS-13 activity assay

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

ADAMTS-13 activity was measured according to the collagen binding assay of Gerritsen et al (1999). Intra-assay and interassay variation coefficients were 8% and 12%, respectively, the lower limit of sensitivity of the assay was 6% of normal protease levels. The lower value of the normal range (46%) was calculated on the basis of the 5th percentile of the distribution in 200 healthy individuals. To evaluate the presence of antibodies neutralizing ADAMTS-13 (inhibitors), plasma samples were incubated at 56°C for 60 min to destroy any residual protease activity. Serial dilutions of test samples in phosphate-buffered saline with 1% bovine serum albumin were mixed with equal volumes of normal pooled plasma and incubated at 37°C for 90 min, and residual ADAMTS-13 activity measured. In the same assay, undiluted normal plasma (taken as 100%) and normal plasma diluted 1:2 and 1:4 (taken as 50% and 25% activity respectively) were also tested. The dilution of patients’ plasma that corresponded to 50% of residual ADAMTS-13 activity was arbitrarily defined as 1 U/ml of inhibitor. This value was also taken as the threshold for the presence of an inhibitor.

Mutation analysis

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

For mutation analysis DNA was isolated from peripheral nucleated blood cells using a salting-out method. Following DNA extraction, all 29 exons and intron–exon boundaries of the ADAMTS13 gene were amplified by polymerase chain reaction (PCR) using previously reported conditions (Levy et al, 2001; Kokame et al, 2002). All PCR fragments were purified using a commercially available kit (PCR96 Cleanup Kit; Millipore, Milan, Italy) and analysed using the ABI PRISM 310 Genetic Analyzer (AB, Applied Biosystems, Monza, Italy). All gene mutations were confirmed by restriction enzyme digestion.

Multimeric analysis of VWF

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

The VWF multimeric pattern was analysed by low-resolution sodium dodecyl sulphate agarose gel electrophoresis using 0.9% low gelling temperature agarose. After electrophoresis, the proteins were transferred to nitrocellulose membranes and stained with peroxidase-conjugated rabbit antibodies against human VWF. VWF multimers were scanned with a densitometer (Scanjet 5200 C; Hewlett Packard, Piscataway, NJ, USA), which resolved the multimers into a series of peaks. Areas under the peaks were calculated by a computer program (Image J; Amersham Pharmacia Biotech, Piscataway, NJ, USA). High molecular weight multimers were arbitrarily defined as the peaks comprising one-third of the length of the gel. The corresponding area was computed and expressed as a percentage of the total area for each gel. Ultralarge VWF multimers were considered to be present when the percentage of high molecular weight multimers was greater than 2 SD above the mean values found in the plasma from 40 healthy individuals.

Clinical manifestations and associated diseases

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

The median age of the 100 patients with a clinical diagnosis of TTP was 37 years (1–85 years), 68 were women and 32 men. Sixty-three patients had a single acute episode of the disease and 10 died in hospital during this episode. There was no particular clinical or laboratory feature that distinguished patients who died from those who survived; four were aged 70 years or more, TTP occurred after bone marrow transplantation in two cases and four cases had not started plasma exchange therapy. In the remaining 37 patients, the disease recurred at least twice after the first acute episode went into remission (defined as normalization of the clinical and laboratory abnormalities diagnostic for TTP for at least one month). None of the patients with recurrent disease died.

In terms of clinical manifestations, 81 patients had central nervous system abnormalities at the time of the first clinical diagnosis, the most prevalent being symptoms of focal ischaemia, seizures and coma. Other frequent symptoms were fever (35 cases), jaundice and gastrointestinal manifestations, such as vomiting, diarrhoea and abdominal pain (25 cases). Only 12 patients had renal abnormalities (oligoanuria and acute renal failure, albuminuria, microscopic haematuria). Bleeding symptoms were not frequent and included epistaxis and menorrhagia (the latter in 7% of the women). Among the 100 patients, 89 had apparently idiopathic TTP, whereas in one the disease was associated with the intake of ticlopidine, in two with allogeneic bone marrow transplantation, in one with pregnancy, and in seven with autoimmune diseases [systemic lupus erythematosus (three), thyroiditis (two) and primary antiphospholipid syndrome (two)]. Plasma exchange was the most commonly used treatment (in 87% of patients), usually in combination with high-dose corticosteroids.

General laboratory findings

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

The most relevant laboratory abnormalities were thrombocytopenia (median platelet count 32 × 109/l, range 10–148 109/l), symptoms of Coombs-negative haemolytic anaemia, such as low haemoglobin concentration (median value 9 g/dl, range 5–12 g/dl), presence of fragmented red cells and high serum LDH levels (median value 2246 IU/ml, range 242–25 050 IU/ml).

ADAMTS-13 activity and inhibitors levels

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

Figure 2 shows the wide range of ADAMTS-13 plasma levels measured in patients. Protease levels were arbitrarily divided into three categories: <10% (severely reduced), 10–46% (moderately reduced) and >46% (normal). Only approximately half of the patients had very low levels of ADAMTS-13 activity (<10%) (Fig 2; Table I), but nearly one-third had normal levels. The clinical and laboratory features and underlying diseases of the patients with normal ADAMTS-13 activity did not differ from those of patients with low levels.

image

Figure 2. Values of ADAMTS-13 activity (open squares, expressed in percentage of average normal plasma) and anti-ADAMTS-13 inhibitor (closed square, expressed in U/ml) plotted according to three arbitrary groups of ADAMTS-13 levels (left panel, <10%; middle panel 11–46%, right panel, >46%). The broken horizontal line indicates the lowest limit of sensitivity of the ADAMTS-13 activity assay (6%). Values below the line are <6%.

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Table I.  Relationship between ADAMTS-13 activity and presence of anti-ADAMTS-13 inhibitors in all patients with acute TTP, and in those with single acute episodes and recurrent TTP.
 ADAMTS-13 activity*
<10%10–46%>46%
  1. The presence of inhibitor is expressed as percentage of the total number of patient for each level of ADAMTS-13.

  2. ND, inhibitor was not tested in patients with normal ADAMTS-13 activity.

  3. Percentage values are given in parentheses.

  4. *Reference range: 46–150%.

All patients (n = 100)48 (48)24 (24)28 (28)
 Inhibitor present (n = 38)33 (87) 5 (13)ND
Single episode (n = 63)29 (46)14 (22)20 (32)
 Inhibitor present (n = 18)16 (89) 2 (11)ND
Recurrent TTP (n = 37)19 (51)10 (27) 8 (22)
 Inhibitor present (n = 20)17 (85) 3 (15)ND

Table I shows that 38 patients had detectable inhibitors (at least 1 U/ml) and Fig 2 shows that inhibitor levels ranged between 1 and 45 U/ml. The great majority of the patients with detectable inhibitor (87%) had severe ADAMTS-13 deficiency (<10%), but five (13%) had moderately reduced ADAMTS-13 activity (ranging from 11% to 25%) (Table I). In the 10 patients who died, the patterns of ADAMTS-13 activity and inhibitor levels were not different from the group as a whole (data not shown).

Table I also shows the relationship between ADAMTS-13 activity and the presence of inhibitors in patients with a single acute episode of TTP compared with those investigated during an acute episode but in the frame of chronic relapsing TTP. In general, the relationship between disease presentation, ADAMTS-13 levels and presence of inhibitors was not different from those observed in the whole group of patients (see Table I).

Mutation analysis

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

Candidate gene mutations were looked for in 15 patients with low ADAMTS-13 levels (<20%) and no inhibitor. Three different mutations of the ADAMTS13 gene were identified in two patients with chronic recurrent TTP and no family history of the disease. One patient, a 20-year-old man from Turkey, was compound heterozygous for two different truncating mutations: a 29 nucleotide deletion in exon 3 (291–319del), not previously reported, and a single base insertion in exon 29 (4143insA), previously described by Schneppenheim et al (2003). The 290–319del and 4143insA mutations lead to premature stops at codons 368 and 1387 respectively. The second patient, a 21-year-old man from Iran, was homozygous for a six nucleotide deletion in exon 23 (2930–2935del), not previously reported. This deletion lead to a substitution of Cys 977 by Trp, and a deletion of two aminoacids, Ala and Arg, at residues 978 and 979 (initiation Met corresponding to +1 codon).

VWF multimeric analysis

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

Among the 40 patients who had samples suitable for VWF multimeric analysis, nine (23%) had ultralarge multimers and three (8%) lacked the largest multimers present in normal plasma, whereas the majority of patients (69%) showed proportions of large multimers within the normal range. Ultralarge VWF multimers were almost always detected in patients with recurrent TTP whereas lack of the largest normal multimers were present only in patients investigated during the acute phase. There were not enough cases with multimeric analysis to evaluate in detail the relationship with ADAMTS-13 levels. However, there were two cases with ultralarge VWF in plasma and normal ADAMTS-13 levels.

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

The main finding of this study is that, in agreement with some investigators (Veyradier et al, 2001; Remuzzi et al, 2002; Studt et al, 2003a; Vesely et al, 2003; Hovinga et al, 2004) but at variance with the original findings of Furlan et al (1998) and Tsai and Lian (1998), the measurement of ADAMTS-13 activity in plasma only helps in the diagnosis of TTP to a limited extent, because not all the patients with overt clinical disease have low plasma levels of the protease. A possible explanation is that our in vitro assay of ADAMTS-13 activity, performed under static conditions and based upon the long incubation of the metalloprotease with the VWF substrate, does not mirror the in vivo flow conditions of the microcirculation, where ultralarge hyperactive VWF is secreted by endothelial cells and then physiologically cleaved into smaller forms by the protease (Dong et al, 2004). However, at least two recent multicentre studies have shown that the assay is useful for the diagnosis of TTP, with acceptable accuracy and reproducibility (Studt et al, 2003b; Tripodi et al, 2004). It is also possible that ADAMTS-13 activity was normal in some patients because regulators of VWF multimer size other than ADAMTS-13 were deficient or dysfunctional (Xie et al, 2001), or because mechanisms unrelated to defective VWF cleavage caused the massive platelet thrombus formation in the microcirculation typical of TTP.

In the 48 cases with severe ADAMTS-13 deficiency, an inhibitor of protease activity was present in 33 (87%), consistent with the frequency of this pathogenetic mechanism reported in other series of similar patients (Veyradier et al, 2001; Studt et al, 2003a; Vesely et al, 2003). An unexpected observation was that an inhibitor was also found, albeit with a smaller frequency, in five patients with moderately reduced ADAMTS-13, after the influence of residual protease in the assay system was ruled out by heating the plasma samples. There are other examples of autoimmune coagulopathies characterized by the incomplete inactivation of the antigen by the autoantibody, e.g. acquired haemophilia caused by anti-factor VIII autoantibodies (Lollar, 2004).

We thought that it was biologically plausible to look for a possible genetic mechanism only in those TTP patients who had low ADAMTS-13 levels and no inhibitor, because the presence or absence of a family history is not particularly informative in recessively inherited disorders, such as TTP. ADAMTS13 gene mutations were found in two of the 15 patients investigated, confirming that the congenital ADAMTS-13 deficiency is rarer than the acquired deficiency. Both patients were born from consanguineous marriages in Muslim populations; in one of them the defect was in the compound heterozygous state and in the other in homozygosity. There was no other clinically affected member in their families.

Several TTP cases had low ADAMTS-13 levels not explained by gene defects. Possible explanations are the presence of non-neutralizing anti-ADAMTS-13 antibodies or other mechanisms that cause an accelerated plasma turnover of the protein (Scheiflinger et al, 2003). There were also two cases characterized by the presence of ultralarge VWF multimers in association with normal protease levels, as previously observed by Remuzzi et al (2002) in two additional cases. This may be explained by the inadequacy of the ADAMTS-13 assay used in this study to accurately measure the failure of the protease to dispose of the highly thrombogenic form of VWF (Dong et al, 2004), or to defects in the regulators of VWF multimer size other than ADAMTS-13 (Xie et al, 2001).

A few clinical considerations can be made on this large series of patients with TTP. In spite of the fact that the referring physicians were of various origin and specialization (haematologists, neurologists, nephrologists, blood bankers, internists) the treatment of choice for TTP – plasma exchange alone or associated with corticosteroids – was implemented in the great majority of patients. Accordingly, mortality was low (10%), within the range of the values reported for patients treated with plasma therapy (Bell et al, 1991; Rock et al, 1991; George, 2000; Zheng et al, 2004). The patients who died had had a single episode of acute disease and none had recurrent disease, but no other clinical and laboratory feature differentiated them from these who survived, except older age, previous bone marrow transplantation and lack of implementation of plasma exchange.

This study, based upon one of the largest series of patients with TTP studied since the introduction of ADAMTS-13 tests, has limitations. First, it is a cross-sectional investigation of plasma samples sent to a tertiary diagnostic centre, with a referral bias that affects the generalizability of the results. Secondly, the diagnosis of TTP was based upon criteria that, albeit objective and pre-established, stemmed from the accuracy in completing a questionnaire. This may have created a misclassification bias, although our adjudication of the diagnosis was performed without knowing the results of ADAMTS-13 assays. Thirdly, the conditions of the study did not enable a detailed follow-up of the patients by means of ADAMTS-13 and inhibitor assays, and so could not answer still incompletely resolved questions, such as the behaviour of ADAMTS-13 during and after plasma therapy, the natural history of inhibitor and the patterns of the anamnestic response (Mori et al, 2002; Knobl et al, 2003; Zheng et al, 2004).

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References

We gratefully acknowledge the following participating centres that provided patient samples and information:

Italian Centres: C. Balduini, San Matteo Hospital, Pavia; U. Occhini, S. Donato Hospital, Arezzo; N. Tannoia, Bari Hospital; R. Vallone, AOG Rummo, Benevento; L. Catani, S. Orsola Hospital, Bologna; C. Cristofalo, A. Perrino Hospital, Brindisi; G. Giuffrida, Ferrarotto Hospital, Catania; P. Spedini, AIO Cremona; G. Gerli, S. Paolo Hospital; L. Gatti, ICP Milan; M. Agnelli, D. Soligo, M. Moroni, IRCCS Maggiore Hospital Milano; E. Rossi, Immunohaematology and Blood Transfusion Service, L. Sacco Hospital of Milan; E. Venegoni, G. Fornaroli, Hospital Magenta; G. Gaidano, AO, Novara; F. Fabris, AO, Padova; M. Rizzuti, S. Carlo Hospital, Potenza; G. Poletti, Ravenna Hospital; P. Accardo, S. Maria Nuova Hospital, Reggio Emilia; R. De Cristofaro, L. Laurenti, Catholic University School of Medicine, Rome; M.T. Pirrotta, Le Scotte Hospital, Siena; F. Marongiu, Brotzu Hospital, Cagliari; C. Cecchini, AO Pesaro; M.C. Bertoncelli, Carità Hospital, Novara.

International Centres: M. Bohm, Klinikum der Johann Wolfang Goethe-Universitat, Frankfurt; C. Mavragani, Department of Pathophysiology, Medical School of Athens, Greece; M. Karimi, Haemostasis and Thrombosis Unit, Haematology Research Centre and University of Medical Sciences, Shiraz, Iran; B. Dolnicar, S. Zver, Department of Haematology Clinical Centre and University Children's Ljubljana; M. Tombuloglu, Ege Universitesi Tip Fakultesi Dahiliye AD Hematoloji BD Bornova, Izmir, Turkey.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patients
  5. Sample collection
  6. ADAMTS-13 activity assay
  7. Mutation analysis
  8. Multimeric analysis of VWF
  9. Results
  10. Clinical manifestations and associated diseases
  11. General laboratory findings
  12. ADAMTS-13 activity and inhibitors levels
  13. Mutation analysis
  14. VWF multimeric analysis
  15. Discussion
  16. Acknowledgements
  17. References
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