Introduction
Thrombotic thrombocytopenic purpura (TTP) is a rare life-threatening thrombotic microangiopathy (TMA) caused by severe deficiency in activity of ADAMTS13, which leads to the accumulation of ultralarge von Willebrand factor (UL-vWF) multimers. UL-vWF multimers bind to platelets and consequently cause platelet aggregation and widespread microvascular ischemia. Acquired TTP is primarily caused by auto-antibodies against ADAMTS-13. It is characterized by a pentad (thrombocytopenia, microangiopathic hemolytic anemia (MAHA), neurological features, fever, and renal involvement). In majority of the cases, no apparent underlying etiology exists, although a genetic link has been identified [
1]. A severe decreased activity of the metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) is present in most cases of TTP, which has been cloned in 2001 [
2]. The mainstay of therapy is plasma exchange (PEX) which can induce remissions in approximately 80% of patients with idiopathic TTP [
3]. Due to urgent requirement for treatment, particularly PEX, to replenish the missing ADAMTS 13 enzyme and to remove the associated antibody, it is important to recognize and diagnose acute TTP as early as possible. Although the “classic pentad” is the typical presentation of the disease, it is neither sensitive nor specific; majority of the patients present without all the five clinical features. Lack of a full pentad may lead to misdiagnosis, and consequently result in risk of suboptimal outcomes, which are potentially avoidable.
In this study, data of patients with acute acquired TTP were collected; laboratory parameters were reviewed; and clinical features, treatments, ADAMTS 13 activities, and outcomes were presented with the purpose of providing a profile for the diagnosis and management of patients with acquired TTP collected in 10 years (2005–2015) in a single center in southeast China.
Materials and methods
Subjects
Patients admitted to the First Affiliated Hospital, College of Medicine, Zhejiang University with a diagnosis of acute acquired TTP from March 2005 to August 2015 were included in the study. Diagnostic criteria included (1) clinical manifestations characterized by thrombocytopenia, microangiopathic hemolytic anemia (MAHA), neurological features, fever, and renal involvement; (2) typical laboratory abnormalities including anemia, thrombocytopenia, schistocytes on blood film, raised lactate dehydrogenase (LDH) with negative Coomb’s test, and normal clotting screen; (3) significantly decreased ADAMTS13 activity; and (4) exclusion of hemolytic uremic syndrome (HUS), disseminated intravascular coagulation (DIC), HELLP syndrome, Evens syndrome, and eclampsia [
4,
5]. Given the factor that ADAMTS13 activity was not measured in all patients due to limited access to validated testing, and normal to moderately reduced plasma ADAMTS13 activity does not completely rule out TTP, criteria number 3 was not a necessity if the patient met the other three criteria.
Clinical data
Clinical data of these cases were collected, reviewing their laboratory parameters, presenting symptoms, treatments, ADAMTS13 levels, and outcomes. Clinical features include symptomatic thrombocytopenia (mucocutaneous hemorrhage, epistaxis, gastrointestinal bleeding, hematuresis, or menorrhagia), MAHA (fatigue, jaundice, anemia, and schistocytes), neurological features (coma, unconsciousness, irrational speech, mental disorder, etc.), fever, and renal involvement (elevated serum creatinine or proteinuria).
Laboratory parameters include cytometry and red cell morphology, reticulocyte, clotting screen, lactate dehydrogenase (LDH), b2 microglobulin, ferritin, hepatorenal function, alkaline phosphatase, thyroid function, and antinuclear antibodies tests.
Measurements of ADAMTS13 activity and antibodies to ADAMTS13
Blood samples were collected from the patients for measurements of ADAMTS13 activity and antibodies to ADAMTS13 before PEX/plasma infusion (PI). ADAMTS13 activity was measured by the First Affiliated Hospital of Soochow University using residual collagen binding assay [
6]. The ADAMTS13 activity of less than 5% was considered as undetectable [
4,
5]. The presence of an inhibitor to ADAMTS 13 was determined by incubating a mixture of patient plasma (1:1) with pooled normal plasma for 1 h at 37 °C and measuring the ADAMTS13 activity by the above residual collagen binding assay. Values<5% are considered as positive for the inhibitor screen.
Assessment of therapeutic effects
Complete remission was defined as reversal of clinical manifestations and thrombocytopenia. Relapse was defined as recurrence of clinical symptoms/laboratory parameters more than 30 days after remission from a previous presentation.
Statistical analysis
Mortality and relapse rate were described using descriptive statistics. Comparisons of measurement data were done with t-test, while comparisons of enumeration data were done with Chi-square test. All statistical analyses were done using the statistical software SPSS version 22.0. P value of<0.05 was considered statistically significant.
Results
Clinical characteristics
Sixty patients with 69 clinical episodes were enrolled in this study; 52 patients presented with their first episodes, and eight patients had two or more episodes. The median age at presentation was 49 (range, 17 to 78) years with a female predominance (male:female ratio of 1:1.60). Dividing the cases into precipitating causes, 50 of 60 (83.3%) cases had no obvious underlying causes and were defined as idiopathic. Other causes included trauma (n = 1), pregnancy (n = 1, with connective tissue disease (CTD)), drug (n = 3), alcohol consumption (n = 2), and infection (n = 3, 1 of whom complicated with CTD). In addition, six of 60 patients (10%) were complicated with CTD.
All the patients were of acute onset, with a median time from initial presentation to diagnosis of 7 days (range, 1 to 35 days). The presenting features were broadly divided into neurological symptoms (including headache, seizures, being slow to respond, mental disorder, numbness of limbs, confusion, and coma), fever, renal impairment, symptomatic thrombocytopenia (petechia, bruising, hematuria, melena, and hematemesis), and hemolysis (jaundice, backache, and soy urine). All of the patients had thrombocytopenia; 59 (98.33%) of the patients had anemia. Of the patients, 46 (78.33%) had neurological symptoms, 18 (30%) had symptomatic thrombocytopenia, 40 (66.67%) had fever, and 10 (16.66%) had renal impairment. Only six (10%) patients had a classic pentad (Table 1). Further division of presentations with neurological features showed that 16 (26.66%) patients of all these cases were admitted in a coma. Compared with survivors, a higher proportion of patients in the death group had hemorrhagic manifestations, renal impairment, and classic pentad (P<0.05; Table 2).
Laboratory parameters
Baseline laboratory parameters on admission were recorded. Low platelet count was present in all the patients; median platelet count was 12 × 109/L (range, 4×109/L–39×109/L) and hemoglobin concentration was 70 g/L (range, 45–147 g/L). Median reticulocyte percentage was 12% (range, 5%–30.7%). Schistocytes were present on film in 53 patients (88.3%) for whom blood film were done, with a median percent of 5% (range, 1%–12%). Fifty-five cases (91.7%) had raised bilirubin levels, with a median value of 58.5 μmol/L (range, 10–206 μmol/L), primarily indicating the underlying hemolysis. Median creatinine level was 87 mmol/L (range, 50–673 mmol/L). Clotting screen was negative in 57 patients, with the other three patients having a slightly prolonged PT or APTT. Elevated LDH was noted in all the patients; the median value of which was 1196 U/L (range, 305–4427 U/L). Besides, 41/43 (96%) of the cases, for which the information was available, had an increased ferritin level; the median level of which was 720.8 ng/ml (range, 274–4000 ng/ml).
ADAMTS13 activity assays and antibody detection
ADAMTS 13 activity levels were analyzed in 43 patients, in which 33 (76.7%) of the patients had a baseline level of<5%. Moreover, detection of antibodies to ADAMTS 13 was performed in 30 cases; 19 (63.3%) cases were positive.
Treatment
In patients with initial presentation, PEX was used in 54 of 60 (90%) patients. PEX was performed once a day in most cases, and occasionally once every other day when plasma was not available. Of the six patients who were not treated with PEX, two patients were treated with PI, an approach that is inferior to PEX, one patient gave up on therapy, and the remaining three patients were administered glucocorticoid alone. In patients with relapsed disease, PEX was used in eight of nine episodes. One patient experiencing a third time relapse of TTP received neither PEX nor PI but was treated with rituximab. Reviewing the total number of PEX administered, the overall median was 3 (range, 1–9), and the overall median volume was 4890 ml (range, 1600–15 100 ml). PI was used together with PEX in 32 patients and alone in two patients. In most cases, PI was administered before PEX, while PEX was being arranged.
In 60 first presenting episode, glucocorticoid was used in 47 cases. Among them, three patients received glucocorticoid alone. In the remaining 44 patients, glucocorticoid was administered in combination with PEX/PI. Furthermore, other immunosuppressive agents were used as combination therapy in six patients (two patients with vincristine, one patient with vincristine and cyclosporine, and three patients with cyclosporin). For relapsed episodes, glucocorticoid was administered in seven of nine cases; immunosuppressant was used in one episode.
Rituximab was documented in four patients with five episodes. In one patient with newly diagnosed TTP refractory to PEX and glucocorticoid, rituximab was administered at a dose of 375 mg/m2 weekly for four successive weeks. For the remaining three patients with relapsed disease, rituximab was given at a dose of 375 mg/m2 in one patient and 100 mg in two patients with three episodes, once a week, for four successive weeks.
Efficacy
Forty-two (70%) patients achieved CR after 3–7 days of treatment. Eighteen (30%) patients died (10 women, 8 men); among whom, seven patients died due to poor response to PEX, two patients received PI alone, and one patient remained untreated for fast disease progression while PEX was being arranged. Eight patients gave up on therapy and were discharged against medical advice. Among all the 60 patients of initial onset, all the patients who were untreated (n = 1) or treated with glucocorticoid alone (n = 3) died. Eight of 12 (66.67%) patients treated with PEX/PI, four of 37 (10.81%) patients treated with PEX/PI+ glucocorticoid, and two of six (33.33%) patients treated with PEX/PI+ glucocorticoid+ immunosuppressant died. The median time from diagnosis to death were 4 days (range, 1–15 days). Details are shown in Table 3.
Among those patients who survived, eight (18.60%) had a relapse after 2–15 months from previous episode. One patient experienced two episodes of relapsed disease and the other seven patients had one relapsed episode as of the endpoint of follow-up. No patient died in relapsed episodes. Compared with survivors, patients who died were much older; had significantly higher lactate dehydrogenase (LDH) and alkaline phosphatase level; and lower platelet count (P<0.05; details are shown in Table 4).
Discussion
TTP is a rare acute life-threatening disease that belongs to the thrombotic microangiopathies (TMA), a group of disorders sharing the common outcome of microvascular ischemia and end-organ dysfunction. The pathogenesis of TTP derives from severe functional deficiency of the Willebrand factor cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) [
7]. Under physiological conditions, ADAMTS13 cleaves ultralarge dimers of VWF (UL-VWF) released from endothelium upon activation or vascular injury [
8,
9]. In the absence of ADAMTS13, UL-VWF is not appropriately cleaved, resulting in the widespread microvascular thrombosis that characterizes TTP [
10]. TTP can be divided into two forms: hereditary TTP, also known as Upshaw–Schulman syndrome (results from biallelic mutation of the encoding gene) and acquired TTP (primarily caused by auto-antibodies against ADAMTS-13). Acquired TTP is a rare disease with an estimated incidence of around two to six per 1 000 000 in the UK and USA, preferentially affecting women [
11,
12]. No accurate data regarding its incidence in China exists and national guidelines for management of TTP due to weak evidence base is lacking. A description and analysis of all the cases of TTP presenting over a 10-year period within a single center in southeast China is presented.
In our study, the majority of patients had an acute onset. In terms of clinical symptoms at presentation, all the patients had thrombocytopenia; a vast majority of patients had microangiopathic hemolytic anemia. Neurological features at presentation were documented in 76.66% of cases, ranging from headache to coma. A classic pentad was seen in only 10% patients, all of whom died during follow-up. In our clinical practice, a diagnosis of TTP should be suspected when a patient presents with thrombocytopenia, microangiopathic hemolytic anemia, or neurological features of unknown reason. Dramatically increased LDH were documented in most cases. In addition, being older, severe thrombocytopenia, and increased AKP at presentation appeared to be associated with increased risk of death.
After clinical assessment and investigations to identify the presence of a thrombotic microangiopathy TMA (i.e., low hematocrit, elevated serum LDH, schistocytes on blood film, etc.), a presumptive diagnosis of TTP can be made. The measurement of plasma ADAMTS13 activity is the most important subsequent investigation for differentiation of TTP from other thrombotic microangiopathies (e.g., atypical hemolytic uremic syndrome (aHUS)). The ADAMTS13 activity assays are neither 100% sensitive nor specific; the results are method dependent, therefore normal to moderately reduced plasma ADAMTS13 activity does not completely rule out TTP [
13,
14]. In patients with plasma ADAMTS13 activity of less than 5%, testing for inhibitory antibodies to ADAMTS13 is essential to distinguish acquired TTP from congenital form [
6]. Congenital TTP is confirmed by mutational analysis of ADAMTS13, the mutation of which may be homozygous or compound heterozygous. While in acquired TTP, antibodies to ADAMTS 13 can be detected [
10].
However, assays exploring ADAMTS13 activity and inhibitor require skill and are offered only by a small number of specialized laboratories given the low incidence of the disease. In our cohort, ADAMTS13 activity was performed in 43 patients; 33 (76.7%) patients had a baseline level of<5%. Inhibitor test was performed in only 30 patients. Of those with inhibitor testing performed, 19 out of 30 patients were positive for the presence of antibodies to ADAMTS13. As a result, it appeared that some of the patients in our cohort were treated for TTP with a suboptimal distinction between TTP and other forms of MAH, acquired and congenital forms. Fortunately, access to validated testing was facilitated in recent years; ADAMTS13 activity and inhibitor/antibody investigations were performed in vast majority of patients after year 2011, subsequently reducing the risk of exposure of patients to potentially ineffective or unnecessary therapies.
PEX remains the mainstay of the management of TTP and should be started as soon as the diagnosis of acquired TTP is made or even suspected. PEX is thought to work by replacing the deficient ADAMTS13 and removing inhibitory auto-antibodies. Given the factor that the results of ADAMTS13 assays may not be immediately available, the decision to commence treatment with PEX must be a clinical decision based on history, physical examination, and laboratory tests. PEX was performed in 62 of 69 (89.9%) episodes in our cohort. Of those in whom PEX was not performed, one patient experienced a third relapse of TTP and was treated with rituximab. Two patients received PI combined with steroids. The remaining four patients suffered from fast disease progression and died or gave up on therapy while PEX was being arranged. Eighteen (30%) patients died in our cohort; the mortality of which was much higher than the reported 4%–14.8% worldwide [
12,
15,
16]. Possible contributors to the high mortality include: (1) eight patients gave up on therapy; (2) large distances from rural hospitals to our hospital which offers specialized services (e.g., PEX) resulted in delay in initiation of PEX due to need of arrangement in interhospital transfer; (3) patients were admitted to the hospital on weekends when apheresis services may be unavailable; and (4) delay in initiation of PEX or insufficient PEX performed due to limited supply of blood products. Reviewing the total number of PEX administered, the overall median was three in our cohort, which was 15.5 in the UK [
12] and 12 in Australia [
17].
In addition to PEX, the other major therapies used in the treatment of acquired TTP are immunosuppressants. Identification of the autoimmune origin of the disease provided a rational basis for wide use of immunosuppressive treatment. Conventional immunosuppressants include steroids, usually started on with PEX, and stronger ones, such as cyclosporine, cyclophosphamide, and vincristine, for patients who fail to respond to first-line treatment. Splenectomy is also an alternative therapy for patients with severe refractory disease. However, the level of proof concerning the efficacy of the aforementioned immunosuppressive agents in the treatment of TTP remains quite low [
13]. In this cohort, corticosteroids were the most commonly used immunosuppressant (in 54 of 69 episodes). Other immunosuppressants (e.g., vincristine, cyclosporine, and cyclosporin) were used in seven of 69 episodes. In recent years, rituximab has been introduced to be an effective adjunct to PEX therapy with encouraging results in refractory and relapsed acquired TTP [
18]. It is now routinely recommended in the acute phase of the disease in patients with a suboptimal response to treatment at a dose of 375 mg/m
2; however, whether it should be proposed as frontline or prophylactic therapy is still debated [
19,
20]. The use of rituximab was documented in four patients in our cohort. Among the four patients, one had a refractory episode of TTP despite PEX and steroids and was treated with rituximab at a dose of 375 mg/m
2 weekly for four successive weeks. The other three patients were treated with rituximab after disease relapsed. For the three patients with relapsed disease, rituximab was given at a dose of 375 mg/m
2 in one patient and 100 mg/m
2 in two patients with three episodes, once a week, for four successive weeks [
21]. All of them achieved favorable outcome and remained long-term remission. It seemed that low-dose rituximab may be as effective as the standard dose for patients with refractory/relapsed acquired TTP, though further prospective clinical trial is required.
Due to improved understanding of the pathology of acquired TTP, an increasing number of novel approaches for the treatment of TTP have entered or will soon enter clinical trial. These new therapies include inhibition of vWf–platelet interaction via anti-Willebrand factor nanobodies, ADAMTS13 substitution by recombinant ADAMTS13, and reduction binding affinity of ADAMTS13 to autoantibodies by alteration of the spacer domain of ADAMTS13. Caplacizumab is a nanobody that targets the A1 domain of VWF preventing the formation of microthrombotic disease by blocking the adhesion of VWF multidimer to platelets. The TITAN study demonstrated that Caplacizumab had a promising value in the treatment of TTP with an acceptable safety profile [
22].
In vitro data suggesting that recombinant ADAMTS13 may be able to overcome the present inhibitor and restore ADAMTS13 protease function [
23]. Mutant ADAMTS13 variants were generated by Jian and colleagues with a decreased autoantibody binding but preserved ADAMTS13 protease function. Though still early in preclinical development, it could potentially be an effective treatment of acquired TTP [
24].
In conclusion, TTP is a hematologic emergency that requires a rapid diagnosis and must be managed as an emergency. This description of diagnosis and treatment of acquired TTP data are from a single center of southeast China. A larger, multicenter study would give further epidemiological information. Efforts are underway to make early diagnosis of TTP, facilitate a more widespread availability of ADAMTS13 activity/inhibitor testing for patients with suspected TTP, as well as optimize a greater access to timely PEX.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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