Clinical trial NCT00713193
Conditions: Thrombotic Thrombocytopenic Purpura
Keywords: Idiopathic TTP Treatment Exacerbation Relapse Plasma Exchange
Sponsor: Ohio State University
Start date: 2007-11-01; Completion date: 2017-09-20
Enrollment: 26 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This research involves the use of immune base therapy as an adjunct to plasma exchange, the present standard of care for thrombotic thrombocytopenic purpura (TTP). Funding source -FDA OOPD

Detailed description:
TTP is a rare blood disorder that causes blood clots to form in the small blood vessels throughout the body, including the kidneys, brain, abdomen, and the heart. Plasma exchange is the standard treatment for TTP. Plasma exchange is a treatment that removes the plasma (the liquid portion of the blood without any cells) from a patient and replaces it with plasma from a donor. With plasma exchange, 90% of patients achieve a remission of the disease. Unfortunately, up to one half of patient will relapse after the plasma exchange has stopped, leading to significant complications and added risks to the patient.

This study randomizes patients to receive either prednisone or cyclosporine as an adjunct to plasma exchange, with the cyclosporine arm being the experimental arm of the study. All patients will undergo plasma exchange but will be randomized to receive either prednisone or cyclosporine as an adjunct to plasma exchange. Previous studies suggested that cyclosporine was superior to prednisone as an adjunct to plasma exchange, and therefore this randomized study attempts to confirm the findings of two previous single institution studies.

Arms: arm 1: Patients in this arm will receive cyclosporine (Neoral) at a dose of 2-3 mg/kg orally as an adjunct to plasma exchange.

All patients initiated daily PEX (one plasma volume), using plasma as the replacement fluid. Patients randomized to the CSA arm received CSA at a dose of 2-3 mg/kg (rounded to the nearest 50 mg increment) divided into a twice daily dosing (Figure 1a). Patients randomized to CSA did not receive steroids, but were permitted to receive hydrocortisone as required for any hypersensitivity reactions to the infused plasma. Daily PEX was continued until a clinical response was achieved, then patients received PEX every other day for 2 additional procedures, with the first day that the platelet count and LDH were normal counting as the first of the 2 procedures. arm 2: Patients in this arm will receive prednisone at a dose of 1 mg/kg as an adjunct to plasma exchange.

All patients initiated daily PEX (one plasma volume), using plasma as the replacement fluid. Patients randomized to the corticosteroid arm received prednisone at a dose of 1 mg/kg/d rounded to the nearest 20 mg increment. Daily PEX was continued until a clinical response was achieved, then patients received PEX every other day for 2 additional procedures, with the first day that the platelet count and LDH were normal counting as the first of the 2 procedures.
Interventions: intervention 1: Cyclosporine intervention 2: Prednisone
Intervention descriptions: intervention 1: 2-3 mg/kg orally in a twice day divided dose for 6 months intervention 2: 1 mg/kg orally, daily for at least 30 days, then tapered over 30 days after achieving remission.

Protocol documents:
Prot_SAP_000.pdf: 
 
- 1 - 
 
 
 
 
 
 
 
 
IRB Approved Version 08.03.2011 
 
 
 
 
 
A Multi-Center, Randomized Study of Cyclosporine or 
Corticosteroids as an Adjunct to Plasma Exchange in Initial 
Therapy of Thrombotic Thrombocytopenic Purpura  
 
 
Coordinating Center:  
 
The Ohio State University  
 
 
 
Protocol Chairman:  
 
Spero Cataland, M.D. 
 
306B Starling Loving Hall 
 
320 W 10th Avenue 
 
Phone: 614-293-2887 
 
Fax: 614-293-7529 
 
Email: spero.cataland@osumc.edu 
 
Protocol Co-Chairman:  
Haifeng Wu, M.D. 
 
(Co-investigator) 
129 Hamilton Hall 
 
 
 
 
 
 
1645 Neil Ave. 
 
 
 
 
 
 
Columbus,OH 43210 
 
 
 
 
 
 
Phone: 614-292-3335 
 
 
 
 
 
 
Fax: 614-292-7072 
 
 
 
 
 
 
Pager: 614- -0832  
 
 
 
 
 
 
Email: Haifeng.wu@osumc.edu 
 
 
 
 
 
 
 
 
Statistician:                   
 
Susan Geyer,  Ph.D.   
 
 
 
 
 
Morehouse Medical Plaza-Pavilion 
 
 
 
 
 
2012 Kenny Rd 
 
 
 
 
 
Associate Professor in Statistics 
 
 
 
 
            Ohio State University   
 
 
 
Study Nurse/Coordinator:  
Leslie Witkoff 
 
 
 
 
 
M414 Starling Loving Hall 
 
 
 
 
 
320 W. 10 Ave. 
 
 
 
 
 
Phone: 293-8183 
 
 
 
 
 
Fax: 293-7529 
 
 
 
 
 
Columbus, OH 43210 
 
Leslie.Witkoff@osumc.edu 
 
 
 


 
- 2 - 
 
 
1.0 
OBJECTIVES 
 
This will be a multi-center, open-label, randomized study that directly compares 
the use of cyclosporine and plasma exchange to corticosteroids and plasma 
exchange as the initial treatment of patients with thrombotic thrombocytopenic 
purpura.  This study has the following objectives:  
 
Primary Objective 
 
1.1  To determine if cyclosporine given individually as an adjunct to plasma exchange 
decreases the rate of exacerbation in patients with TTP compared to 
corticosteroids given as an adjunct to plasma exchange. 
 
Secondary Objectives 
 
 
1.2   To compare the CSA and PE treatment arm to the corticosteroid and PE treatment 
arm with respect to: 
 
 exacerbation rate within 30 days post-PE 
 clinical response rate 
 number of exchange procedures to achieve clinical response 
 relapse rate (recurrent disease >30 days after last PE)  
 safety data for CSA as an adjunct to PE. 
 the effects of corticosteroids vs. cyclosporine on ADAMTS13 antigen, activity, 
and ADAMTS13 antibody concentration and inhibitor titers using serial 
measurements both during and after discontinuation of study drug 
 the VWF multimeric patterns after initial therapy and throughout longitudinal 
follow-up between the two treatment arms  
 the laboratory biomarker(s) that are prognostic for TTP exacerbation and the risk 
of relapse 
 
2.0 
BACKGROUND 
2.1 
Clinical Presentation and Therapy of TTP 
 
 
There have been tremendous advances in our knowledge and understanding of 
the pathophysiology of TTP since Drs. Amorosi and Ultmann characterized the pentad 
of findings (fever, microangiopathic hemolytic anemia, thrombocytopenia, neurologic, 
and renal findings) in a 1966 review of all 271 previously reported cases.(1)  Despite 
early attempts at therapy which included corticosteroids, splenectomy, and heparin, 
TTP was almost uniformly fatal with 90% of patients dying from the disease.  Following 
observations of clinical improvement with plasma-based therapy, clinical trials soon 
demonstrated an 80-90% remission rate. (2)  Rock et al reported one of the only 
randomized clinical trials in TTP and were able to demonstrate the superiority of 


 
- 3 - 
therapeutic PE over simple plasma infusion in the acute treatment of TTP.(3) Based 
upon this study, PE is considered the standard of care of acute TTP.   
 
 
Despite the dramatic improvement in outcome with PE, complications related to 
the central venous catheter (infection, hemorrhage, and pneumothorax) and the plasma 
infusion (hypersensitivity, volume depletion, and transmission of infection) may be 
associated with significant morbidity and mortality.  Howard et al recently updated their 
experience with PE for TTP at the Oklahoma Blood Institute, reporting roughly a third of 
patients experienced significant complications from PE and a 2.4% mortality rate.(4)  
More importantly, approximately 50% of patients with idiopathic or acquired TTP will 
suffer a recurrence of the disease after tapering plasma exchange, requiring repeated 
exposure to the risks of PE therapy.(5)  These historical data were confirmed by our 
cohort study of corticosteroids and PE in which 60% of patients required the re-initiation 
of plasma-based therapy within 30 days of discontinuing PE therapy.(6) 
 
2.2 
Advances in the Pathophysiology of TTP 
The earliest observations by Amorosi and Ultmann from post-mortem examinations 
described microthrombi in multiple organs of the body, including the heart, kidneys, 
adrenal glands, and the brain.(1)  The etiology of the microthrombi was not known, but 
the findings were fitting with the clinical presentation and course of the patients.  In 
1982, Moake et al were able to demonstrate that some patients with TTP had unusually 
large multimeric forms of vWF, similar to those stored in vascular endothelial cells and 
platelets, but typically absent from normal plasma.(7, 8)  Given the finding that these 
forms disappeared with the acute presentation of TTP and reappeared in patients after 
remission, it was hypothesized that these highly active multimers of vWF were central to 
the pathogenesis of the microthrombi seen in TTP.  It was postulated that a vWF 
“depolymerase” was responsible for the conversion of unusually large multimers of vWF 
to the smaller multimers that are usually found in the circulation.(9)  In this scenario, the 
unusually large multimers of vWF would spontaneously react with platelets resulting in 
the platelet thrombi in the microvasculature.  It was also hypothesized that an antibody 
inhibitor of the “depolymerase” could account for the unusually large multimers seen in 
the plasma based upon reports of patients with chronic relapsing TTP being controlled 
with immunosuppressive therapy.(10)  The subsequent identification of a specific 
protease(11, 12) that was shown to cleave purified human vWF as well as the finding 
that patients with TTP had deficiencies of protease function further strengthened the 
hypothesis.  Furlan et al went on to describe both a familial form of TTP who had a 
constitutional deficiency, as well as a non-familial form due to an inhibitor of the vWF-
cleaving protease.(13)  This protease, now known as ADAMTS13, was recently purified 
and the gene localized to the long arm of chromosome 9.(14, 15)  
 
2.3 
ADAMTS13 Activity and Inhibitors in Idiopathic TTP 
Initial reports correlating ADAMTS13 activity with the clinical diagnosis of acquired TTP 
showed greater than 70% of patients with idiopathic TTP to have a severe deficiency of 
protease activity (< 5%) with the majority having demonstrable antibody inhibitors of the 


 
- 4 - 
protease.(13, 16, 17)  Autoantibody inhibitors of ADAMTS13 are now recognized to be 
central to the pathophysiology of idiopathic TTP, as well as patients with pregnancy 
associated TTP and TTP secondary to an autoimmune disease (SLE) (18, 19).  
Additionally, persistent or recurrent antibody inhibitors of ADAMTS13 have been 
associated with recurrences of TTP as well as a chronic relapsing form of the disease, 
giving more validity to the hypothesis of targeting the antibody inhibitor of ADAMTS13 
(20-22).  In their review of 142 consecutive patients with TTP from the Oklahoma TTP-
HUS Registry, Vesely et al described an inverse relationship between ADAMTS13 
activity and both exacerbations (recurrent thrombocytopenia requiring the resumption of 
PE less than 30 days from the cessation of PE) and relapses of TTP that was most 
striking in patients with a severe ADAMTS13 deficiency. (21)   Of the 18 patients with a 
severe deficiency of the protease, all but one had a demonstrable antibody inhibitor.  
Therefore, although patients are able to achieve a clinical remission with PE, a 
persistent antibody inhibitor of ADAMTS13 likely is a factor contributing to higher rates 
of exacerbation and relapse in patients with idiopathic TTP.  Similar data was reported 
by Zheng et al, showing that 3 of 4 patients with severe ADAMTS13 deficiency and high 
titer inhibitory antibodies had a chronic relapsing course of their disease.(23)  In these 4 
patients PE did not result in any detectable plasma ADAMTS13 activity or a consistent 
reduction in the inhibitor titer.  It is important to note that the risk of relapse in patients 
with persistent inhibitors is not uniform, as not all patients with persistent inhibitors are 
destined to relapse.(24)  Recent data has also suggested that a severe deficiency of 
ADAMTS13 protease function alone may not always be sufficient to trigger an acute 
episode of TTP.(25)   
 
2.4 
Immunotherapy of Idiopathic TTP 
There is an obvious rationale for the role of immunosuppressive therapy in patients with 
idiopathic TTP, both as an adjunct to PE therapy as well as a prophylactic therapy 
preventing the recurrence of disease.  In theory, PE would serve as a “supportive 
therapy”, physically removing the inhibitor and inducing remission, while at the same 
time allowing CSA, a “disease-modifying therapy”, time to exert its effect.  An effective  
immunosuppressive therapy could inhibit the production of the antibody inhibitor of 
ADAMTS13, resulting in shorter courses of PE, decreased exacerbations and relapse 
rates, and decreased exposure to the potential risks associated with plasma based 
therapy.  Many cytotoxic and immunosuppressive agents have been reported to be 
effective in relapsed and refractory TTP, including: corticosteroids, cyclophosphamide, 
vincristine, cyclosporine, and rituximab.(22, 23, 26-38)  Unfortunately, there has been 
very little good, prospective data with which to accurately judge the effectiveness of a 
particular therapy without confounding factors.  Rituximab has been reported to be 
effective in refractory TTP, as well as being effective as prophylactic therapy in patients 
with recurrent inhibitors and a chronic relapsing course of TTP.(22)  Rituximab though 
may have significant drawbacks including the need for intravenous administration, the 
potential for infusional toxicity and infectious complications.  The issue of the active 
removal of rituximab when combined with PE therapy also remains to be addressed.  
Advantages with both corticosteroids and cyclosporine include their ability to be 


 
- 5 - 
administered orally, and in the case of cyclosporine, the ability to be administered 
concurrently with PE without significantly affecting cyclosporine concentration.(39)   
 
 
 
2.5 
Corticosteroids 
Corticosteroids are routinely been used and recommended(40) as an adjunct to PE, but 
until recently no prospective studies have addressed their efficacy as an adjunct to PE.  
Among the series of patients reported by Zheng et al, four patients with undetectable 
ADAMTS13 activity and high titer inhibitors of the protease were followed serially. Two 
were treated with corticosteroids as an adjunct to plasma exchange. Both showed no 
sustained improvement in ADAMTS13 activity or inhibitor titers during the follow-up 
period.  Data from our prospective cohort study of corticosteroids as an adjunct to PE 
also showed that the addition of corticosteroids to PE did not reduce the exacerbation 
rate compared to historical data from the Oklahoma TTP registry where corticosteroids 
are not routinely used.(6, 21, 41) 
 
2.6 
 Clinical Outcome- Concurrent Corticosteroids and PE as Initial Therapy 
 A total of 12 patients with idiopathic TTP were treated with corticosteroids and PE.  
Patients received prednisone at a dose of 1 mg/kg/day concurrently with daily plasma 
exchange therapy which was continued until remission was achieved (platelet count 
>150 x 109/ul).  PE was then tapered and discontinued with the prednisone tapered over 
the next 4 weeks.  Ten of 12 patients achieved remission after a median of 6.5 daily 
exchanges, but exacerbations of TTP  occurred in 6/10 (60%), comparable to the 50% 
exacerbation rate from the Oklahoma Blood Institute where steroids were not routinely 
used. (Table 1)  More importantly, there was no significant improvement in ADAMTS13 
activity at remission nor suppression of the inhibitor measured within a week of tapering 
PE.  Given these data, we then began to study alternative forms of immunosuppressive 
therapy as an adjunct to PE. 
 
 
 
 
 
 
 
 
 
 
 
 
3.0 
Rationale for Concurrent Cyclosporine and Plasma Exchange in TTP 
Cyclosporine has been reported to be effective in cases of relapsed or refractory 
TTP.(26, 30, 31, 33)  Our group has published a series of 3 patients with early 
  Clinical Data               
Remission 
10/12 
Med. Days to Remission 
6.5 (range, 4 to 10) 
Exacerbation  Rate 
6/10 (60%) 
Laboratory Data  
Med. Pre-Treatment ADAMTS13 
<1% (range, <1 to 5) 
Med. Remission ADAMTS13 
4% (range, <1 to 72)  
 Med. Pre-Treatment Inhibitor (ug/ml) 
 632 (range 188 to 3,574) 
Med. Remission Inhibitor (ug/ml) 
 788 (range 144 to 8,113) 
 
Table 1.  
 
Clinical and  
Laboratory Data 
From 
Steroid and  
Plasma 
Exchange- 
Treated Patients   
 


 
- 6 - 
recurrences of TTP treated with cyclosporine alone, without the need for plasma 
exchange or infusion.(42)  In addition to the clinical improvement seen, all 3 patients 
had significant increases in ADAMTS13 activity and suppression of the autoantibody 
inhibitor that correlated with a clinical remission of their disease.   The therapeutic effect 
of CSA is thought to be related to its ability to inhibit the activation of genes necessary 
for the B-cell “helper” function (IL-4 and CD 40 ligand) of T-cells and T-cell proliferation 
(IL-2) required for the sustained production of high-affinity antibodies and memory B-
cells to perpetuate a significant and sustained antibody response.(43)  There is also 
increasing evidence that CD4+/CD25+ T-regulatory cells may be important in the control 
of pathologic immune responses in autoimmune hematologic disorders such as 
autoimmune hemolytic anemia and immune thrombocytopenic purpura (44-47).  Low 
dose therapy with cyclosporine has also been shown to promote the development of 
CD24+/CD25- T-regulatory cells in an animal model, providing an additional rationale for 
the use of CSA concurrent with PE.    
 
3.1 
Concurrent Cyclosporine and PE for Exacerbations and Refractory TTP   
Ten patients with exacerbations or refractory TTP after their initial therapy received CSA  
concurrent with the resumption of PE.(48)  All 10 patients were treated with concurrent 
CSA (2-3 mg/kg in a twice daily divided dose) and PE.  CSA was continued for a total of 
6 months and then discontinued.  Nine of the 10 patients achieved remission after a 
median of 11 daily exchanges (range, 5-45), with only 1/9(9%) suffering an 
exacerbation after tapering PE.  Median follow-up for the entire cohort is 31 months 
(range, 12 to 41).  Eight of the 10 patients maintained a continuous remission 
throughout the planned 6 months of CSA and then discontinued therapy.   
 
3.2 
Concurrent Cyclosporine and PE as Upfront Therapy  
Building on the findings of our initial study that suggested a clinical benefit for the 
addition of CSA to PE, we initiated a study of concurrent CSA and PE as the upfront 
therapy of idiopathic TTP.  Eleven of 12 patients achieved remission after a median of 5 
daily exchanges.  Median follow-up for the entire cohort was 16 months (range, 1 to 25).  
None of the 10 evaluable patients achieving remission have suffered an exacerbation, a 
statistically significant difference compared to the corticosteroid treated patients (0% v. 
60%, p=<0.05).{Cataland, 2006 #375} Seven of the 10 patients maintained a continuous 
remission throughout the planned 6 month course of CSA, with one recently enrolled 
patient having only completed 5 months of CSA to date.  Two patients suffered a 
recurrence of TTP while still taking CSA after 3 and 4 months of CSA therapy 
respectively, with one of the patients relapsing 2 weeks after a 50% dose reduction of 
CSA as mandated by the study for an increase in the serum creatinine to 1.5 mg/dl 
(range, 0.9-1.4 mg/dl).  Further analysis of this patient revealed his CSA concentration 
to be sub-therapeutic, arguing that his mild renal insufficiency was not related to CSA 
toxicity, but rather may have been related to his underlying thrombotic microangiopathy.  
These data from both cohort studies suggest that the use of CSA as an adjunct to PE 
resulted in a clinically and statistically significant reduction in the exacerbation rate 


 
- 7 - 
compared to concurrent corticosteroids and PE, preventing the need for re-exposure to 
the potential risks associated with PE therapy.   
 
3.3 
Combined Analysis: ADAMTS13 Activity, Antigen and Inhibitor After      
Concurrent CSA and PE 
Patients on both cohort studies (exacerbation/refractory study and upfront study) of 
CSA and PE were followed serially to study the effect of CSA on ADAMTS13 activity, 
antigen, and inhibitor concentration and correlated with clinical outcome data.  
Complete ADAMTS13 and inhibitor data obtained serially for all CSA and PE treated 
patients during and after the 6 month period of CSA therapy is shown graphically in 
Figure 1.  Although not randomized, these data suggest that cyclosporine improved 
ADAMTS13 activity via the suppression of the inhibitory IgG antibody.  Although it could 
be argued that the improvement in ADAMTS13 activity could have resulted from PE 
therapy, the significant delay before the improvement in the ADAMTS13 activity argue 
that the CSA resulted in the suppression of the inhibitory antibody and the improvement 
in ADAMTS13 activity.  Furthermore, the gradual decline seen after the discontinuation 
of CSA also support the hypothesis that it was the CSA and not the delayed effect of PE 
that resulted in the steady improvement in ADAMTS13 activity seen in this cohort of 
patients.   
0
5
10
15
20
25
30
35
40
45
50
0
2
4
12
24
36
44
60
90
108 132
ADAMTS13%
0
500
1000
1500
2000
2500
3000
IgG Inhibitor (ug/ml)
ADAMTS13%
IgG Inhibitor Conc.(ug/ml)
 
Figure 1.  Serial determinations of the mean ADAMTS13% activity and inhibitor concentration 
throughout clinical follow-up both during and after therapy with cyclosporine.  The arrow at the 
top of each graphs represent the duration of therapy with cyclosporine.  
 
 
4.0 
Toxicity of Combined Analysis of CSA and PE  
Overall therapy with concurrent CSA and PE was well tolerated.  Two patients treated 
with concurrent cyclosporine and PE had increases of the serum creatinine to 1.5 mg/dl 


 
- 8 - 
(normal < 1.4 mg/dl) after two and 4 weeks of cyclosporine therapy; both patients had 
their dose of cyclosporine decreased by 50% with subsequent normalization of the 
serum creatinine.  Although this was presumed to be related to the cyclosporine, the 
concentrations of the drug obtained at the same time were not above the therapeutic 
range, raising the possibility that this mild increase in the serum creatinine may be 
related to the renal effects of TTP.  Mild gastrointestinal toxicity was transiently 
experienced by 2 patients as well but did not require a decrease in the dose or 
frequency of the CSA therapy 
 
5.0 
 Patient Selection  
 
5.1 
Inclusion of Women and Minorities.  
 
Entry to this study is open to both men and women ages 18 and older, of all 
racial and ethnic subgroups.  This study will be conducted in accordance with the 
International Conference on Harmonisation and the Helsinki Guidelines. 
 
5.2 
Eligibility Criteria.  
 
 
5.2.1 Patients with a clinical diagnosis of idiopathic TTP as defined by a 
microangiopathic hemolytic anemia and thrombocytopenia (<100 x 103) 
 
 
 
5.2.2 Additional components of the pentad (fever, renal and neurologic 
abnormalities) need not be present. 
 
 
5.2.3 Additional explanations for the microangiopathic changes including DIC 
and malignancy should be excluded. 
 
 
5.2.4 Patients with pregnancy associated TTP will be permitted on this 
therapeutic trial if the child is delivered prior to the initiation of therapy for 
TTP.  However, female patients that are breastfeeding and are unwilling to 
discontinue breastfeeding at the time of enrollment will be excluded from 
this study (see section 5.3.5) 
 
 
5.2.5 Patients with a previous diagnosis of TTP are eligible to be enrolled 
provided they meet eligibility criteria and have not been treated for  TTP in 
the past 30 days. 
 
 
5.2.6 Given the potential for nephrotoxicity with CSA, all patients must have a 
serum creatinine of < 2.5 mg/dl prior to enrollment 
 
 
5.3 
Exclusion Criteria.  
 
5.3.1 In light of concern for the prompt initiation of PE, all patients with 
suspected TTP may be enrolled on this trial. If it is subsequently found 
that the patient does not meet enrollment criteria, they will be removed 
and their spot replaced for study purposes.  Patients removed from the 


 
- 9 - 
study after enrollment will continue to be followed longitudinally for 6 
months to be monitored for safety and will be included in the safety 
database. 
 
 
 
5.3.2 Patients with TTP clinically categorized as secondary to stem cell 
transplant and solid organ, bloody diarrhea associated, malignancy 
associated, and drug associated will not be enrolled on this therapeutic 
study. 
 
5.3.3 Incarcerated patients will be excluded from the study due to the inherent 
difficulties in maintaining close follow-up for study purposes in patients 
who are incarcerated.   
 
5.3.4 Any patients already being treated chronically with corticosteroids or 
cyclosporine and taking these at the time of their presentation will be 
excluded from this study.  
 
5.3.5 Female patients that are pregnant or breastfeeding and are unwilling to 
discontinue breastfeeding at the time of enrollment will be excluded from 
this study 
 
5.3.6 Patients taking any medications contraindicated in combination with CSA 
that cannot be safely discontinued will be excluded from this study.  A 
complete list of contraindicated medications is provided in Appendix 4. 
 
5.4 
ENROLLMENT/REGISTRATION OF PATIENTS 
 
5.4.1 Patients must give written, informed consent prior to enrollment on this 
study.  Informed consent may be obtained by the protocol chairman, the 
principal investigator at one of the additional sites, or one of their 
designees. 
 
 
 
It is not uncommon for patients to have mental status changes related to 
their diagnosis of TTP (typically reversible with therapy) that may prevent 
them from being able to provide informed consent.  If the principal 
investigator, co-investigator, or research study nurse do not feel the 
patient is able to provide informed consent, the study consent may be 
signed by the patient’s legally authorized representative to enroll the 
patient in the study. 
 
 
If the patient’s legally authorized representative signs the consent, the 
patient must be then re-consented with the “re-consent form” if/when the 
patient regains the ability to provide informed consent.   
 
5.4.2 Given concerns for the need to rapidly initiate, PE therapy should not be 
withheld while awaiting enrollment to this study.  To allow sufficient time to 
evaluate for eligibility and obtain informed consent, patients will be 
permitted to be enrolled within 48 hours after the first PE procedure. 


 
- 10 - 
 
5.4.3 At the time of enrollment on this protocol, patients must be registered by 
phone with the study coordinating office at Ohio State University, the lead 
institution for this study. Contact information for all study personnel, 
protocol chairman, and co-investigators is detailed in section 15.0 of the 
protocol.  
 
6.0 
Randomization of Patients 
 
 
 
6.1 
Patients will be randomized at the time of registration/enrollment on this 
clinical trial via contact with the study nurse/coordinator at the time of the 
initial registration of the subject.  The study statistician (Dr. Susan Geyer) 
will conduct the randomization and relay this information to the study 
nurse who will be in contact with the treating/enrolling physician so that 
the appropriate medication (prednisone or cyclosporine) may be 
immediately started with PE.   
 
 
 
 
 
7.0  
Treatment Plan: Initial Therapy 
 
7.1 
Therapeutic Plasma Exchange-Both Study Arms 
 
7.1.1 Plasma exchange will be started as soon as the diagnosis of TTP has 
been made. (section 5.2.1).  As it is important to institute therapy as soon 
as possible, patients will not have therapy delayed while obtaining 
informed consent.   
 
7.1.2 Plasma exchange will be performed daily with one plasma volume 
exchanged using plasma as the replacement fluid until a complete 
remission is obtained (see response criteria 8.0).  
  
7.1.3 Patients will receive either prednisone or cyclosporine orally as an adjunct 
to PE depending on the outcome of randomization (see sections 7.2-7.3 
below).  Therapy with these adjuncts will be initiated concurrent with and 
continue throughout plasma exchange therapy as described below.   
 
7.1.4 After a complete remission (see response criteria 8.0) has been attained 
patients will have one exchange performed every other day for a total of 2 
more exchanges, with the first day that the platelet count and LDH are 
normal counting as the first of the two exchanges. 
 
7.2 
Corticosteroid Study Arm 
 
7.2.1 At the time of diagnosis, patients will be started on daily prednisone 
therapy at a dose of 1 mg/kg/day (rounded off to the nearest 20 mg 
multiple) concurrently with daily PE.  Patients unable to take oral 
corticosteroids will be switched to an equivalent intravenous corticosteroid 
until they are able to take oral medications.  Patients may receive 
intravenous corticosteroids at any time to urgently treat reactions to the 


 
- 11 - 
infused plasma and for prophylaxis against future reactions prior to the 
next TPE.   
 
7.2.2 Prednisone should be continued at full dose for 30 days after the last PE 
procedure. Subsequently, patients should decrease the dose by 50% each 
week over 4 consecutive weeks, starting on day 31. All corticosteroids 
should be discontinued completely by the end of the 4 week taper 
(approximately 8 weeks total of corticosteroid therapy). 
 
7.3 
Cyclosporine (Neoral) Study Arm 
 
 
7.3.1 As not all brands of cyclosporine can be used interchangeably due to the 
potential for inconsistencies in cyclosporine drug concentrations, 
physicians will use the Neoral brand of cylcosporine whenever possible.  If 
an additional brand must be used, the patient must stay on that same 
brand of cyclosporine throughout the 6 month course as the differing 
brands cannot be used interchangeably.   
 
7.3.2 At the time of diagnosis, patients will be started on daily cyclosporine 
(Neoral) therapy at a dose of 2-3 mg/kg/day (rounded to the nearest 50 
mg increment) concurrent with daily plasma exchange.  Doses will be 
administered in the capsule form.  The total dose will be divided into twice 
daily dosing. For consistency and standardization of cyclosporine levels 
throughout the study, doses of Neoral (cyclosporine) should be given at 8 
AM and 8 PM each day.  Neoral (cyclosporine) should be taken on an 
empty stomach, and patients should not eat for at least 30 minutes after 
their dose of Neoral (cyclosporine).  Cyclosporine will be continued for a 
total of six months of therapy and then discontinued.   
 
7.3.3 Patients with renal insufficiency at presentation will have the dose of CSA 
modified to minimize any potential added nephrotoxicity of CSA.  Patients 
with a serum creatinine of 2.5 mg/dl or greater will not begin therapy with 
CSA until the serum creatinine has fallen below this threshold.  Patients 
with a serum creatinine of 1.5-2.5 mg/dl will have the CSA dose 
decreased by 50% until the serum creatinine has normalized (< 1.5 mg/dl).  
After renal function as measured by the serum creatinine has normalized 
patients will have the dose increased to the intended dose of 2-3 
mg/kg/day. 
 
7.4 
Management of Cyclosporine Dosing and Concentrations  
 
7.4.1 The optimal therapeutic concentration of CSA in TTP is not known.  For 
the purpose of this study, the target CSA concentration will be identical to 
the accepted therapeutic CSA concentration at each institution(100-400 
ng/ml at Ohio State University) when used as immunosuppressive 
therapy.  As described below, dose adjustments of CSA will not be made 
to reach this goal, but rather the dosing will only be changed for patients 
with supratherapeutic concentrations or a decline in renal function.  Only 


 
- 12 - 
trough concentrations should be used to adjust the CSA dose.  If patients 
have already taken their CSA on the morning of the CSA concentration 
measurements, dose adjustments should only be made in clinical toxicity 
is present (hypertension. anxiety, diarrhea, emesis).   
 
7.4.2 Cyclosporine levels (trough) will be monitored after the third day of 
therapy, weekly for the first 4 weeks of therapy and then monthly while 
they are taking CSA to monitor drug levels and minimize any potential 
nephrotoxic effects (see section 9.2, Laboratory Studies During Plasma 
Exchange) Patients should not take their CSA prior to having the CSA 
levels drawn, but should take their scheduled dose after their blood is 
drawn that day in order to check trough CSA levels.  Trough levels of CSA 
should be obtained at 8 AM, at the same time the morning dose of CSA is 
to be given for hospitalized patients.  CSA levels will then be obtained at 
each follow-up visit to monitor drug concentrations to minimize the 
potential for CSA toxicity.  The CSA dose will not be increased based on 
these levels, only decreased or held as a response to potentially toxic 
levels of CSA.   
 
7.4.3 Patients with trough CSA concentrations above the upper limit of normal 
will have the dose altered as described below and levels checked again in 
3 days (see Appendix 2).  Subjects with trough cyclosporine concentration 
of 401-500 ng/ml should decrease the dose of cyclosporine by 25% and 
recheck the trough concentration in 3 days.  Subjects with cyclosporine 
concentrations of 501-600 ng/ml should decrease the dose by 50% and 
recheck the concentration in 3 days.  Subjects with cyclosporine 
concentrations greater than 600 ng/ml should hold any further 
cyclosporine doses until the levels are rechecked in 3 days.  If the 
cyclosporine concentration is now less than 600 ng/ml, a 50% dose 
reduction should be performed with future dose adjustments made 
depending on future cyclosporine concentration determinations as 
described above.  Although there is a theoretical concern regarding the 
effect of daily plasma exchange on CSA levels, it has been reported that 
plasma exchange had no significant effect on measured CSA levels.(39)  
 
7.5 
Dose Adjustments During Cyclosporine Therapy and Renal Insufficiency 
 
          7.5.1 Patients who subsequently develop renal insufficiency during the 6 month 
course of CSA (serum creatinine >1.5, but < 2.5 mg/dl) will have the dose 
decreased by 50% (see Appendix 2).  The dose will be returned to the 
same planned dose (2-3 mg/kg) after the serum creatinine has returned to 
normal (<1.5 mg/dl).  If patients do not tolerate the resumption of CSA to 
the full dose (recurrent increase in the serum creatinine, high CSA levels), 
patients should be maintained on the dose level that was able to achieve a 
therapeutic CSA levels without renal toxicity.  Patients with a serum 
creatinine greater than 2.5 mg/dl will have the CSA held, and restarted 
only when the serum creatinine is < 2.5 mg/dl, with the change in dosing 
schedule the same as described above. 


 
- 13 - 
 
8.0 
Clinical Response Criteria 
 
8.1 
Complete Remission 
 
A patient will be deemed to be in remission if all of the following criteria have 
been met within 4 weeks from enrollment and maintained after the tapering of 
plasma exchange: 
 
- 
platelet count >150,000 
- 
normalization of LDH  
- 
a decreased or unchanged serum creatinine compared to the creatinine 
measurement obtained at the time of enrollment. 
- 
clinical 
improvement 
in 
neurologic 
findings/deficits 
without 
the 
development of new neurologic findings/deficits by exam or radiographic 
studies. 
 
8.2 
Partial Response 
 
 
8.2.1 Patients showing improvement (increase in platelet count and a decrease 
in the LDH, but not meeting the full criteria for complete remission) should 
continue concurrent CSA or prednisone and plasma exchange therapy for 
up to 4 weeks.  If at that time patients still have not met the criteria for 
remission, patients will be considered to have been unable to achieve 
remission but will remain on this protocol for the purposes of data and 
clinical sample collection and for safety monitoring, but may be treated at 
the discretion of their treating physician.   
 
 
8.3 
Refractory Disease 
 
 
8.3.1 Patients not showing signs of improvement (partial or complete response) 
or benefit with daily plasma exchange and CSA therapy after 1 week of 
continuous therapy should be considered to have refractory disease.  
These patients will remain on this protocol for the purposes of data and 
clinical sample collection and for safety monitoring, but may be treated at 
the discretion of their treating physician and will be considered to have 
failed therapy. 
 
8.4 
Exacerbation/Relapse Determination  
 
8.4.1 Criteria to determine recurrences of disease and the need for re-initiation 
of therapy with plasma exchange will be the same as those to diagnose 
TTP (section 5.2.1).  Patients requiring the reinstitution of plasma-based 
therapy (plasma exchange or infusion) within the first 30 days after 
discontinuing PE will be considered to have an “exacerbation” of their 
TTP.   
 


 
- 14 - 
8.4.2 Patients with a recurrence of TTP at a time point greater than 30 days 
from the end of plasma exchange therapy will be considered to have a 
“relapse” for the purposes of this study. 
 
8.5 
Treatment after Recurrences of TTP 
 
8.5.1 Patients suffering exacerbations of TTP as described in the protocol 
(Section 6.4) after treatment on the corticosteroid arm of this study may 
then be treated with CSA concurrent with the resumption of plasma 
exchange at the same dose and schedule as described in the CSA arm of 
this randomized trial, assuming that they still meet enrollment criteria 
(including the serum creatinine).  Patients will also discontinue the 
corticosteroids on the same day that CSA is initiated.  Patients treated 
with CSA and PE for exacerbations of TTP will then be prospectively 
followed as described below (Section 9.3 – 9.5), but will be analyzed 
independently from the patients treated with CSA and PE as the upfront 
therapy. 
 
8.5.2 Patients suffering a relapse (recurrence greater than 30 days after the last 
exchange procedure) whether during CSA or after completing 6 months of 
CSA will continue to be followed on this study for the purposes of 
gathering follow-up data, but may be treated at the discretion of the 
treating physician.  
9.0 
Clinical Studies 
9.1 
 Laboratory Studies at Enrollment  
 
9.1.1 The initial laboratory tests will be obtained at the time of enrollment on this 
study. Laboratory tests will include (see Appendix 1): 
 
·CBC (complete blood count), platelet count and evaluation of the   
peripheral smear  
·chemistry panel (Na, K, Cl,CO2, Cr, BUN, Ca, and glucose), total and     
direct    bilirubin 
 ·LDH (lactate dehydrogenase), haptoglobin 
 ·PT /PTT /Fibrinogen 
 ·Direct antiglobulin test (DAT) 
 ·liver function tests (AST,ALT, GGT, Alkaline Phosphatase) 
9.1.2 We will collect at least 100 cc plasma and 10 cc/2 teaspoons of whole 
blood prior to beginning of the plasma exchange for following laboratory 
investigations:  
  
 
·ADAMTS13 activity  
 
 
·ADAMTS13 inhibitor assay  


 
- 15 - 
 
 
·ADAMTS13 antigen level  
 
 
· 
9.2 
Laboratory Studies During Plasma Exchange  
9.2.1 During plasma exchange therapy, the following labs will be obtained  
 
daily (see Appendix 1):  
 
·CBC with a differential and platelet count 
 
·chemistry panel (Na, K, Cl, CO2, Cr, BUN, Ca, and glucose) with LDH 
 
9.3 
Laboratory Studies During Plasma Exchange-Cyclosporine Monitoring 
9.3.1 For patients randomized to the CSA arm of the study, CSA concentrations 
should be obtained on the 3rd day of PE, and then weekly thereafter as 
long as they remain in the hospital concurrent CSA and PE  (see 
Appendix 1). 
9.3.2 Samples for CSA levels should be obtained in the morning, prior to the 
first dose of CSA in order to obtain trough levels of CSA (section 7.4.2). 
 
9.4 
Outpatient/Remission Follow-up and Correlative Laboratory Studies 
 
9.4.1 After discharge from the hospital, patients will be seen weekly in the 
outpatient      clinic for the first month.  Patients will then be seen monthly 
for the next 6 months, regardless of which arm of the study they were 
randomized to (see Appendix 1).   
 
9.4.2 The following laboratory studies will be obtained at each follow-up visit: 
 
 
 
·CBC with a platelet count  
 
·chemistry panel (Na, K, Cl,CO2, Cr, BUN, and glucose), total and 
direct bilirubin  
 
 
 
·LDH  
 
·cyclosporine concentration (patient should be told not take their 
CSA dose until after the blood has been drawn to ensure that we 
are accurately measuring trough drug levels   
 
9.4.3 In addition patients will have peripheral blood samples (10cc/2 teaspoons) 
obtained at the same intervals for:  
 
 
 
·ADAMTS13 activity  
 
 
 
·ADAMTS13 inhibitor assay  
 
 
 
·ADAMTS13 antigen level  


 
- 16 - 
 
 
 
· 
 
 
 
-ADAMTS13 antibody (IgG) concentration 
 
 
 
-VWF multimeric analysis 
 
 
9.5 
Outpatient Visits 
 
9.5.1 After discharge from the hospital, patients will be seen weekly in the 
outpatient clinic for the first month.  Patients will then be seen monthly for 
the next 6 months, regardless of which arm of the study they were 
randomized to.  After six months of continuous follow-up patients will then 
be seen every 3 months for the next 3 years to monitor the course of their 
disease and obtain laboratory follow-up studies (see Appendix 1) 
 
10.0 Safety Monitoring During Study 
 
10.0.1 To ensure the safety of all patients enrolled on this study careful      
monitoring and follow-up for all patients will be an important aspect of this 
study.  At the time patients begin therapy with plasma exchange and 
either CSA or prednisone, all patients will be hospitalized and monitored 
for both response to therapy as well as any potential toxicities.  Laboratory 
studies performed daily will monitor the clinical response to therapy, but 
will also monitor for the earliest signs any other end-organ toxicities 
(Section 9.1 – 9.4). In the CSA-treated patients the potential for renal 
toxicity is the primary concern, but patients on both arms will be monitored 
carefully for this as well as other potential toxicities through daily 
laboratory as well as daily clinical evaluations.  After discharge, patients 
will continue to be monitored closely in the same manner with laboratory 
and clinical evaluations at regular intervals as described in Section 9.5 
and Appendix 1 of the study protocol. 
 
10.1 Definition of Adverse Events/Severe Adverse Events 
 
10.1.1 Adverse events as defined in the International Council on Harmonisation 
Guideline for Good Clinical Practice are described as: “an untoward 
medical occurrence in a patient or clinical investigation administered a 
pharmaceutical product that does not necessarily have a causal 
relationship with this treatment” (ICH E6:1.2).   
 
10.1.2 A serious adverse event is an adverse event that: 
 
 
-is fatal 
 
-is life threatening (places the subject at immediate risk of death) 
-results in inpatient hospitalization or prolonged existing hospitalization 
-results in persistent or significant disability/incapacity 


 
- 17 - 
-is a congenital anomaly/birth defect 
-other significant hazard 
 
10.2 Reporting of Adverse Events 
 
 
10.2.1 The principal investigator at each site is responsible for reporting each 
adverse event to the Protocol Chairman or the study nurse/coordinator 
coordinating site (Ohio State University Hospital).  Contact information is 
provided in section 16.0 of the study protocol.  Adverse events must be 
reported verbally and faxed to the coordinating site within 7 days of the 
occurrence using the supplied “Adverse Event” form (see Appendix 3).  All 
serious adverse events must be communicated to the coordinating site 
verbally within the first 24 hours of the occurrence of the event.  The 
“Adverse Event” form (see Appendix 3) must also be completed and 
returned to the coordinating site within 7 days of the occurrence of the 
event.  Using these mechanisms of rapid reporting we will be able to 
rapidly evaluate and minimize the potential risks to patients from the 
occurrence of these events.  Any significant adverse events that 
potentially would affect other enrolled patients and those yet to be enrolled 
will be communicating to all participating sites and investigators in a 
written format and via teleconference if warranted. 
 
 
 
10.3 Follow-up of Adverse Events 
 
 
10.3.1 Patients experiencing an adverse event will continue to be followed and 
carefully monitored until the resolution of the adverse event.  This applies 
whether the patient continues on study or is removed from the study for 
any reason. 
 
 
 
11.0 Sample Procurement  
 
11.0.1 As stated in section 9.0, both plasma and blood samples will be taken as a 
part of this protocol.  Theses samples will be obtained at the same time 
that standard clinical samples or procedures are being performed so 
patients will not be exposed to additional venipunctures or procedures by 
their participation in this study.  The plasma to be studied is the byproduct 
of the plasma exchange procedure and would otherwise be discarded.  
Instead of discarding the entire plasma sample, 100 cc will be collected for 
study as is described in the protocol.   
 
11.1 Sample Storage and Handling 
  
11.1.1 At the time of enrollment and registration with the coordinating institution, 
arrangements will be made for the biologic samples to be delivered to the 
laboratory of Dr. Haifeng Wu for processing and analysis.  Samples will be 


 
- 18 - 
labeled with a unique identification number so that the biological data 
obtained may be correlated with the patient’s clinical course by the 
investigators.  This will help ensure patient confidentiality, as the 
investigators will be the only people to know the patient name and medical 
record number that correspond with the identification number.  Samples to 
be used for future study will be frozen at -80C and also be stored in the 
laboratory of Dr. Wu.  Laboratory studies unique to this protocol, including: 
ADAMTS13 activity, antigen level, and inhibitor assays will be performed 
in Dr. Wu’s laboratory.     
 
11.2 Sample Utilization 
 
11.2.1 As our understanding of the pathophysiology of TTP increases, we will 
likely discover additional proteins or molecules that play a role in the 
initiation or the progression of TTP.  It is for this reason that we will bank 
samples for future study. The patient samples held in storage will be kept 
indefinitely or until the sample is completely utilized for correlative studies.  
The patient samples will only be studied at the Ohio State University and 
will not be shared with any other researcher for study without prior 
approval of the IRB.  The subject may withdraw consent at any time and 
the sample remaining and any data will be destroyed to the extent that is 
possible.  Patients also will not receive any financial gain from the 
research associated with the tissue sample research or its applications.   
 
12.0 Risks Associated with Testing of Patient Samples 
 
12.1 A breach of confidentiality is the most serious risk which patients may be 
exposed to regarding to the testing of biologic samples.   Confidentiality 
will be maintained by labeling the patient samples with only an 
identification number and not their name or medical record number.  By 
using this approach, patient samples may be tied to clinical information by 
the investigators but will preserve the confidentiality of patients.  Only the 
principal investigator, co-investigator, and the study nurse/coordinator will 
have access to both the study number and the corresponding patient 
identification information.  Clinical information or laboratory data used for 
publication will not identify patients in any way that could potentially 
breach patient confidentiality.   
 
13.0 ADAMTS13 Assays and ADAMTS13 Inhibitor Assays 
 
 
13.1 Novel SELDI-Based ADAMTS13 Assay 
 
Dr. Wu’s laboratory has invested tremendous efforts to validate and 
standardize the tests for ADAMTS13 activity and inhibitory titer to meet 
the requirements set forth by the College of American Pathologists (CAP) 
for clinical testing. Both tests are done using a SELDI-TOF mass 
spectrometer. Validation studies were performed to evaluate test 
accuracy, test precision, reportable range, and test linearity.  For example, 


 
- 19 - 
test accuracy was partly evaluated by testing a set of unknowns and 
correlating the results with those from the Coagulation Reference 
Laboratory at the BloodCenter of Wisconsin (kindly supported by Dr. Ken 
Friedman).  Excellent correlation was achieved between SELDI-TOF and 
the FRET assays for ADAMTS13 (activity and inhibitor titer). ADAMTS13 
activity and inhibitor titer assayts are now offered as clinical tests at 
OSUMC and are performed under SOPs with QC reagents (normal and 
abnormal) included in each run to ensure test accuracy. Longitudinal 
evaluation of QC samples is performed to monitor the test reproducibility 
and analytical performance of the instrument.  
In late 2005, Dr. Wu’s laboratory started using the SELDI-TOF mass 
spectrometry-based method to determine ADAMTS13 activity in patient 
samples (49).  This new test offers a rapid result within 4 hours and has 
excellent test reproducibility (6, 42, 49, 50). In 2008, Dr. Wu laboratory 
modified the SELDI-TOF assay to now detect as low as 0.5% of 
ADAMTS13 activity in clinical samples (51). With this improvement we 
now can more precisely measure ADAMTS13 activity in most samples 
with extremely low activity.  This in turn provides a dataset with 
quantifiable values in >95% of TTP samples to help for statistical analysis.   
 
 
13.2  Laboratory Method for ADAMTS13 Antigen and Antibody  (IgG) 
Concentrations 
 
Quantification of ADAMTS13 antigen and ADAMTS13 autoantibody (IgG) 
will be measured using commercially available ELISA kits from American 
Diagnostica Inc. (IMUBIND® ADAMTS13 ELISA and ADAMTS13 
Autoantibody ELISA). These two kits have been validated in our laboratory 
and used for several studies (6, 42, 49, 50).  The reference ranges for 
both kits were determined using healthy donors (n=41) recruited from the 
local population. The results for the autoantibody ELISA are reported as 
micrograms of IgG antibody and for ADAMTS13 antigen reported as 
nanograms of ADAMTS13 protein per milliliter of patient plasma 
respectively. 
 
13.3 Laboratory Method for ADAMTS13 Inhibitor Titer Determination 
 
In order to evaluate ADAMTS13 inhibitory titer, we set up a SELDI-TOF based 
test for the determination of inhibitory titer as Bethesda units (BU). BU in 
each sample is defined as the dilution of patient plasma that neutralizes 
50% of ADAMTS13 activity in an equal volume of pooled normal plasma 
(PNP).  This method is similar to the standard laboratory procedure for the 
determination of BU for clotting factors that requires 1 hour incubation 
after mixing patient plasma (or dilute) with PNP(52).13.4 Laboratory 
Method for VWF multimeric analysis: 
VWF multimeric analysis will be performed in Biomarker Reference 
Laboratory at the Ohio State University Medical Center. Dr. Wu is the 


 
- 20 - 
director of this clinical laboratory. The cost for this test will be paid from 
research fund under this clinical trial study.  
 
 
 
14.0 Statistical Analysis 
 
14.1  Statistical Considerations-Sample Size Determination  
 
 
 
 
 
For the study of the primary endpoint (treatment success rate), the sample 
size required is calculated based on the data from our previous publications. 
In these previous studies that looked separately at CSA/PE in 10 patients and 
at corticosteroids/PE in another 10 patients, we saw promising results that led 
to our hypothesis that CSA/PE treatment for TTP patients results in greater 
treatment response. In those studies, the primary endpoint was the proportion 
of patients who had TTP exacerbation, which is a more stringent endpoint that 
does not include other types of treatment failures such as refractory patients 
and/or death within 30 days post-PE. In the previous published pilot studies, 
we showed that 6/10 corticosteroid-treated patients exacerbated versus 0/10 
CSA-treated patients. Combined with experience with subsequently treated 
patients, we observed an overall treatment success rate of 80% and 83% in 
refractory vs. de novo TTP patients, respectively, treated with CSA+PE 
versus 33% in those treated with corticosteroids+PE.  
 
  
In terms of our primary endpoint of treatment success rate, we have 
designed a phase III trial that compares the proportion of treatment successes 
in each arm. Specifically, this study design will have at least 80% power to 
determine a significant difference in the treatment success rates between the 
arms if the true rates are 50% vs. 80%, assuming a significance level of 0.05 
and a two-sided test. This reflects a clinically meaningful difference, and the 
data from our previous studies and experience support it as a feasible 
difference to detect. We plan to enroll a total of 72 patients to this trial (36 to 
each arm) to test the null hypothesis that there is no difference in treatment 
success rate between these treatments.  
 
 
 
14.2 Analysis Plan 
 
 
 
 
Patients will be followed for 30 days after their last PE procedure unless a 
treatment failure has been documented prior to that timepoint. The treatment 
success rates will be calculated for each of the treatment arms along with 
their 95% confidence intervals. These proportions will be compared between 
treatment arms using Mantel-Haenszel chi-square test to accommodate the 


 
- 21 - 
stratification factor (recurrent vs. de novo TTP event). Statistical significance 
will be concluded based on a significance level of 0.05.  
 
  
This study includes an interim analysis and formal “look” at the data 
after 24 patients have been accrued (12 to each arm), where the null 
hypothesis will be rejected if the calculated p-value comparing the two 
observed success rates for the treatment arms is <0.0002 (based on the Lan-
DeMets alpha spending function and O’Brien-Fleming boundary calculation). 
We will also evaluate safety and tolerability data for each of the arms at the 
interim analysis, and these data will be reviewed not only by the study team 
but by the DSMB.  
 
  
Through this study we also planned an internal pilot study to be 
completed after 12 patients have been accrued (6 to each arm) in an effort to 
obtain information on the feasibility of accrual and treatment follow-up for the 
endpoints; as an internal pilot study, all patients will be included in the overall 
trial and thus included in the interim and final analyses unless serious 
logistical issues were identified that would introduce bias by their inclusion. 
We reached the accrual point for this internal pilot study and no such 
logistical issues were identified; therefore, all patients will be included in the 
overall study. With so few patients, this internal pilot was used for 
assessment of feasibility; the early data were not formally evaluated and did 
not require a re-evaluation of our assumptions for the study design described 
above.  
 
 
 
 
  
Additionally, we will evaluate several secondary clinical endpoints 
through this trial, including: 1) exacerbation rate within 30 days post-PE; 2) 
clinical response rate; 3) the number of PE procedures required to achieve a 
clinical response; 4) relapse rate; and 5) safety and tolerability of each of the 
adjuvant treatments to PE.  Each of the rates will be calculated assuming 
that these endpoints are binomially distributed, and 95% confidence intervals 
generated. For the exacerbation rate endpoint, we will only include those 
patients who have achieved clinical response (i.e. excluding refractory 
patients) and compare these rates between the two arms. We will have at 
least 80% power to detect a difference if the true exacerbation rates are 55% 
vs. 90% for this subset analysis (not adjusting for multiple comparisons); 
however, this comparison is distinctly ancillary to our primary clinical interest 
of achieving treatment success in all patients treated versus only keeping 
responding patients from experiencing TTP exacerbation. We will also 
evaluate relapse as a time-to-event variable, where Kaplan-Meier methods 
will be used to assess time to relapse from last plasma exchange in 
responding patients. The number of exchange procedures required to 
achieve clinical response will be assessed between arms using Poisson 
regression. Safety and tolerability data will be summarized by type of 
adverse event with frequencies calculated and compared between arms. In 
addition to these planned quantitative analyses, we will also use graphical 


 
- 22 - 
analyses to assess patterns and differences in these factors between the 
treatment arms. 
 
 
14.3  Statistical Considerations-Correlative Laboratory Studies  
 
 
 
The correlative biomarker outcomes described above will be descriptively 
summarized across and within treatment arms, and patterns in these changes 
in biomarkers and how they relate to treatment success, relapse, and TTP 
exacerbation will be explored graphically. For the study to determine the 
effects of corticosteroids and cyclosporine given individually on serial 
measurements of ADAMTS13 biomarker profiles and VWF multimeric 
patterns after their initial therapy and throughout longitudinal follow-up, we will 
evaluate these biomarkers in relation to the clinical outcomes of interest. For 
treatment success rates, we will evaluate baseline levels of these biomarkers 
to identify if there are any associations between these biomarkers and how 
these patients respond to treatment. These will first be explored graphically, 
also denoting those patients in the two different treatment arms. Logistic 
regression for whether or not the patient had a treatment success will be 
modeled with these baseline biomarkers as well as treatment arm.   
 
  
In addition, we will compare the changes for each biomarker between 
the two groups. TTP exacerbations and relapses will be modeled separately. 
Both of these events are preceded by a period of clinical response where 
patients exhibit resolution of clinical signs and normal laboratory values.  
However, as stated above, there are important differences that warrant 
modeling TTP exacerbation and TTP relapses independently. First, the 
frequencies of specimen collection are different between the observation 
periods that lead to the exacerbation and relapses. The patients in the period 
of initial clinical response are monitored weekly, while the patients in the 
sustained clinical remission are followed quarterly. Secondly, the underlying 
pathophysiology for exacerbation and relapse are likely to be different. The 
disease exacerbation probably represents a continuation of an incompletely 
resolved disease while relapses of TTP most likely represent a de novo 
episode of TTP. As a result, the biomarkers that predict TTP exacerbation and 
relapse are likely to be different.  
 
  
To examine the potential of the 4 indices (ADAMTS13 activities, 
ADAMTS13 antigen levels, ADAMTS13 (IgG) autoantibody levels, and 
antibody inhibitor titers) to predict the recurrences of TTP, we will use the 
longitudinal data in two ways by separating indices obtained during the 
sustained clinical remission when they are measured every three months 
(quarterly) from those indices measured during the initial clinical response 
when patients are monitored weekly.  The data for the relapse analysis will 
come from all quarterly samples collected from qualified study subjects during 
sustained remission.  The outcome of interest will be whether or not the 


 
- 23 - 
individual experiences a relapse of TTP during the three months following the 
date the measurements are taken. We anticipate obtaining a total of >400 
observations that will correspond to approximately >16 events of relapse and 
other quarterly measurements (n ~ 400) without relapses. For analysis of 
exacerbation in initial clinical remission, the outcome of interest will be 
whether or not the individual experiences an event of exacerbation in the 
week following the measurement.  The weekly samples in initial clinical 
remission from the same group of study subjects will be analyzed. We 
anticipate a total of about 200 weekly data points, corresponding to about >16 
measurements with events of exacerbation and 200 measurements without 
events in the week following measurement. We will use the logistic regression 
model to analyze all 4 indices independently as well as to evaluate their 
interacting effects.  
 
  
Since each individual has several observations, possibly with and 
without relapse or exacerbation, this would induce a special correlation 
structure. For this reason, we will introduce random effects into the model (53-
55). The covariates corresponding to gender, age, race, and treatment 
modalities (e.g. CSA vs. corticosteroids) will also be evaluated in the model.  
Additionally, various variance-covariance structures will be investigated. The 
Generalized Estimation Equation will also be used if necessary if our specific 
covariance structures lead to un-interpretable results.  Since the number of 
predictor variables is relatively small, a standard traditional model selection 
procedure will likely be feasible to study the reach of the predictors as well as 
their interacting effects. Various model selection criteria such as Ra2, Mallows 
Cp, AIC, and BIC will be used to evaluate the putative models. In the case 
where samples are not available on all subjects at consistent timepoints, we 
will utilize the more flexible growth curve modeling approach to identify 
changes over time in the biomarker on the incidence and time to relapse as 
well as TTP exacerbation.  
  
VWF multimer patterns form a relatively complex dataset. We will 
dissect VWF patterns into several parameters that will all give rise to a 
continuous measurement. This will be the density of VWF multimers in 
bands 1 through 15 and the area above band 15. All data will be 
normalized by the reference plasma included in each gel. For investigating 
the relationship between ULVWF (area above band 15) and relapse in the 
quarter immediately following, a logistic regression analysis as described 
will be performed. Specifically, the response variable will be 1 or 0, 
depending on whether relapse has occurred or not. The explanatory 
variable will be the ULVWF value at the beginning of the quarter-interval, 
with a random effect also included to account for correlations among the 
different response variable observations over time in a given patient. The 
effects of other covariates will be included in the model and adjusted for as 
necessary.  
 
15.0 Costs of the Study 


 
- 24 - 
 
15.1 Given the fact that patients all patients will be receiving standard therapy 
for TTP (plasma exchange therapy), either the patient or their insurance 
carrier will be responsible for all of the medical expenses associated with 
the treatment of their TTP.  The costs associated with the experimental 
laboratory studies will be paid by the study researchers and will not be 
passed on to patients or their insurance carriers.  Patients enrolled on this 
study will not incur any additional expenses as a result of enrolling on this 
protocol.   


 
- 25 - 
 
 
16.0 Contact Information for Study Personnel: 
 
 
Protocol Chairman: 
 
Spero R. Cataland, M.D. 
 
306B Starling Loving Hall 
 
320 W. 10th Ave. 
 
Columbus,OH 43210 
 
Phone: 614-293-2887 
 
Fax: 614-293-7529 
 
Pager: 614-346-5457 
 
Email: spero.cataland@osumc.edu 
 
 
Protocol Co-Chairman: 
 
Hafeng M. Wu, M.D. 
 
129 Hamilton Hall 
 
1645 Neil Ave. 
 
Columbus,OH 43210 
 
Phone: 614-292-3335 
 
Fax: 614-292-7072 
 
Pager: 614- -0832  
 
Email: haifeng.wu@osumc.edu 
 
 
 
Study Nurse/Coordinator at Ohio State University: 
Leslie Witkoff, R.N. 
M414 Starling Loving Hall 
320 W. 10 Ave. 
Phone: 293-8183 
Fax: 293-7529 
Columbus, OH 43210 
 
 
 
 


 
- 26 - 


 
- 27 - 


 
- 28 - 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Yes
No
Continue at Same Dose 
1. Decrease dose by 25% 
 
2. Recheck level in 3 days 
 
1. Decrease dose by 50% 
 
2. Recheck level in 3 days 
 
401-500 ng/ml 
501-600 ng/ml 
> 600 ng/ml 
Appendix 2. Algorithm for dose adjustment of cyclosporine for both elevated drug levels and renal 
insufficiency 
 
*reduce cyclosporine dose if not already done. If previously dose reduced and stable serum creatinine, 
continue at same dose.  If cyclosporine was previously held and the serum creatinine is now > 1.5 mg/dl 
but < 2.5 mg/dl , restart at 50% of planned dose (1.0-1.5 mg/kg). 
 
Serum creatinine <1.5mg/dl 
Yes
No
Continue at Same Dose. 
If previous dose reduction, 
resume full dose 
cyclosporine 
(2.0-3.0 mg/kg) 
50% cyclosporine dose 
reduction*, recheck 
serum creatinine 
 
Serum Creatinine:  
>1.5 mg/dl, but 
< 2.5 mg/dl 
Serum Creatinine:  
>2.5 mg/dl 
 
Hold cyclosporine, 
recheck serum creatinine 
 
Cyclosporine concentration  
(trough level) 
 
< 400 ng/ml 
1. Hold cyclosporine, 
recheck level in 3 days 
 
2. Restart at 50% dose 
reduction if next CSA level 
< 600 ng/ml 


 
- 29 - 


 
- 30 - 
Appendix 4.  Drugs Contraindicated in Combination with Cyclosporine (Neoral) 
 
 
 
 
 
 
Bosentan (probable) 
 
Alfalfa (probable)  
 
Atorvastatin (probable)  
 
Black Cohosh (probable)  
 
Caspofungin (probable)  
 
Cerivastatin (probable)  
 
Colchicine (probable)  
 
Cyclophosphamide (probable)  
 
Etoposide (probable)  
 
Felodipine (probable)  
 
Itraconazole (probable)  
 
Lovastatin (probable)  
 
Nafcillin (probable)  
 
Octreotide (probable)  
 
Orlistat (probable)  
 
Posaconazole (probable)  
 
Pyrazinamide (theoretical)  
 
Red Yeast Rice (probable)  
 
Rifabutin (theoretical)  
 
Rifampin (probable)  
 
Rosuvastatin (probable)  
 
Simvastatin (established)  
 
St John's Wort (established)  
 
Sulfinpyrazone (established)  
 
Tacrolimus (theoretical)  
 
Voriconazole (established) 
 
 
 
 
 
 
 
 
 
 


 
- 31 - 
 
References 
 
 
 
1. 
Amorosi EL, Ultmann JE. Thrombotic Thrombocytopenic Purpura: report of 16 cases and review 
of the literature. Medcine 1966;45:139-59. 
2. 
Bell WR, Braine HG, Ness PM, et al. Improved survival in thrombotic thrombocytopenic purpura-
hemolytic uremic syndrome. Clinical experience in 108 patients.[see comment]. New England Journal of 
Medicine 1991;325(6):398-403. 
3. 
Rock GA, Shumak KH, Buskard NA, et al. Comparison of plasma exchange with plasma infusion 
in the treatment of thrombotic thrombocytopenic purpura. Canadian Apheresis Study Group. N Engl J Med 
1991 Aug 8;325(6):393-7. 
4. 
Howard MA, Williams LA, Terrell DR, et al. Complications of plasma exchange in patients 
treated for clinically suspected thrombotic thrombocytopenic purpura-hemolytic uremic syndrome. 
Transfusion 2006;46:154-55. 
5. 
George JN, Vesely SK, Terrell DR. The Oklahoma Thrombotic Thrombocytopenic Purpura-
Hemolytic Uremic Syndrome (TTP-HUS) Registry: a community perspective of patients with clinically 
diagnosed TTP-HUS. Semin Hematol 2004 Jan;41(1):60-7. 
6. 
Cataland SR, Jin M, Ferketich AK, et al. An evaluation of ciclosporin and corticosteroids 
individually as adjuncts to plasma exchange in the treatment of thrombotic thrombocytopenic purpura. Br J 
Haematol 2007 Jan;136(1):146-9. 
7. 
Moake JL, McPherson PD. Abnormalities of von Willebrand factor multimers in thrombotic 
thrombocytopenic purpura and the hemolytic-uremic syndrome. Am J Med 1989 Sep;87(3N):9N-15N. 
8. 
Moake JL, Rudy CK, Troll JH, et al. Unusually large plasma factor VIII:von Willebrand factor 
multimers in chronic relapsing thrombotic thrombocytopenic purpura. New England Journal of Medicine 
1982;307(23):1432-5. 
9. 
Furlan M, Robles R, Solenthaler M, et al. Deficient activity of von Willebrand factor-cleaving 
protease in chronic relapsing thrombotic thrombocytopenic purpura. Blood 1997;89(9):3097-103. 
10. 
Byrnes JJ, Moake JL. Thrombotic thrombocytopenic purpura and the haemolytic-uraemic 
syndrome: evolving concepts of pathogenesis and therapy. Clin Haematol 1986 May;15(2):413-42. 
11. 
Furlan M, Robles R, Lamie B. Partial purification and characterization of a protease from human 
plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 1996 May 
15;87(10):4223-34. 
12. 
Tsai HM. Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its 
conformation and requires calcium ion. Blood 1996 May 15;87(10):4235-44. 
13. 
Furlan M, Robles R, Solenthaler M, et al. Acquired deficiency of von Willebrand factor-cleaving 
protease in a patient with thrombotic thrombocytopenic purpura. Blood 1998 Apr 15;91(8):2839-46. 
14. 
Levy GG, Nichols WC, Lian EC, et al. Mutations in a member of the ADAMTS gene family cause 
thrombotic thrombocytopenic purpura.[comment]. Nature 2001;413(6855):488-94. 
15. 
Zheng X, Chung D, Takayama TK, et al. Structure of von Willebrand factor-cleaving protease 
(ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura. J Biol Chem 
2001;276(44):41059-63. 
16. 
Tsai HM, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic 
thrombocytopenic purpura. N Engl J Med 1998;339(22):1585-94. 
17. 
Veyradier A, Obert B, Houllier A, et al. Specific von Willebrand factor-cleaving protease in 
thrombotic microangiopathies: a study of 111 cases. Blood 2001 Sep 15;98(6):1765-72. 
18. 
Veyradier A, Meyer D. Thrombotic thrombocytopenic purpura and its diagnosis. J Thromb 
Haemost 2005 Nov;3(11):2420-7. 
19. 
Sadler JE, Moake JL, Miyata T, et al. Recent advances in thrombotic thrombocytopenic purpura. 
Hematology (Am Soc Hematol Educ Program) 2004:407-23. 
20. 
Zheng XL, Kaufman RM, Goodnough LT, et al. Effect of plasma exchange on plasma 
ADAMTS13 metalloprotease activity, inhibitor level, and clinical outcome in patients with idiopathic and 
nonidiopathic thrombotic thrombocytopenic purpura. Blood 2004 Jun 1;103(11):4043-9. 


 
- 32 - 
21. 
Vesely SK, George JN, Lammle B, et al. ADAMTS13 activity in thrombotic thrombocytopenic 
purpura-hemolytic uremic syndrome: relation to presenting features and clinical outcomes in a prospective 
cohort of 142 patients. Blood 2003;102(1):60-8. 
22. 
Fakhouri F, Vernant JP, Veyradier A, et al. Efficiency of curative and prophylactic treatment with 
rituximab in ADAMTS13-deficient thrombotic thrombocytopenic purpura: a study of 11 cases. Blood 2005 
Sep 15;106(6):1932-7. 
23. 
Zheng XL, Kaufman RM, Goodnough LT, et al. Effect of plasma exchange on plasma 
ADAMTS13 metalloprotease activity, inhibitor level, and clinical outcome in patients with idiopathic and 
non-idiopathic thrombotic thrombocytopenic purpura. Blood 2004 Feb 24. 
24. 
Studt JD, Hovinga JA, Radonic R, et al. Familial acquired thrombotic thrombocytopenic purpura: 
ADAMTS13 inhibitory autoantibodies in identical twins. Blood 2004 Jun 1;103(11):4195-7. 
25. 
Banno F, Kokame K, Okuda T, et al. Complete deficiency in ADAMTS13 is prothrombotic, but it 
alone is not sufficient to cause thrombotic thrombocytopenic purpura. Blood 2006 Apr 15;107(8):3161-6. 
26. 
Bachman WR, Brennan JK. Refractory thrombotic thrombocytopenic purpura treated with 
cyclosporine. Am J Hematol 1996 Jan;51(1):93-4. 
27. 
Chemnitz J, Draube A, Scheid C, et al. Successful treatment of severe thrombotic 
thrombocytopenic purpura with the monoclonal antibody rituximab. Am J Hematol 2002 Oct;71(2):105-8. 
28. 
Gutterman LA, Kloster B, Tsai HM. Rituximab therapy for refractory thrombotic 
thrombocytopenic purpura. Blood Cells Mol Dis 2002 May-Jun;28(3):385-91. 
29. 
Fakhouri F, Teixeira L, Delarue R, et al. Responsiveness of thrombotic thrombocytopenic purpura 
to rituximab and cyclophosphamide. Ann Intern Med 2004 Feb 17;140(4):314-5. 
30. 
Hand JP, Lawlor ER, Yong CK, et al. Successful use of cyclosporine A in the treatment of 
refractory thrombotic thrombocytopenic purpura. Br J Haematol 1998 Mar;100(3):597-9. 
31. 
Itala M, Remes K. Excellent response of refractory life-threatening thrombotic thrombocytopenic 
purpura to cyclosporine treatment. Clin Lab Haematol 2004 Feb;26(1):65-7. 
32. 
Kierdorf H, Maurin N, Heintz B. Cyclosporine for thrombotic thrombocytopenic purpura. Ann 
Intern Med 1993 Jun 15;118(12):987-8. 
33. 
Nosari A, Bernuzzi P, Corneo R, et al. Late response to cyclosporine in refractory thrombotic 
thrombocytopenic purpura. Int J Hematol 2002 Oct;76(3):284-6. 
34. 
Pasquale D, Vidhya R, DaSilva K, et al. Chronic relapsing thrombotic thrombocytopenic purpura: 
role of therapy with cyclosporine. Am J Hematol 1998 Jan;57(1):57-61. 
35. 
Stein GY, Zeidman A, Fradin Z, et al. Treatment of resistant thrombotic thrombocytopenic 
purpura with rituximab and cyclophosphamide. Int J Hematol 2004 Jul;80(1):94-6. 
36. 
van Ojik H, Biesma DH, Fijnheer R, et al. Cyclosporin for thrombotic thrombocytopenic purpura 
after autologous bone marrow transplantation. Br J Haematol 1997 Mar;96(3):641-3. 
37. 
Sallah S, Husain A, Wan JY, et al. Rituximab in patients with refractory thrombotic 
thrombocytopenic purpura. J Thromb Haemost 2004 May;2(5):834-6. 
38. 
Yomtovian R, Niklinski W, Silver B, et al. Rituximab for chronic recurring thrombotic 
thrombocytopenic purpura: a case report and review of the literature. Br J Haematol 2004 Mar;124(6):787-
95. 
39. 
Balogun RA, Sahadevan M, Sevigny J, et al. Impact of therapeutic plasma exchange on 
cyclosporine kinetics during membrane-based lipid apheresis. Am J Kidney Dis 2001 Jun;37(6):1286-9. 
40. 
Allford SL, Hunt BJ, Rose P, et al. Guidelines on the diagnosis and management of the thrombotic 
microangiopathic haemolytic anaemias. Br J Haematol 2003 Feb;120(4):556-73. 
41. 
Coppo P, Wolf M, Veyradier A, et al. Prognostic value of inhibitory anti-ADAMTS13 antibodies 
in adult-acquired thrombotic thrombocytopenic purpura. Br J Haematol 2006 Jan;132(1):66-74. 
42. 
Cataland S, Jin M, Zheng X, et al. An evaluation of cyclosporine alone for the treatment of early 
recurrences of thrombotic thrombocytopenic purpura. J Thromb Haemost 2006;4(5):1162-64. 
43. 
Ho S, Clipstone N, Timmermann L, et al. The mechanism of action of cyclosporin A and FK506. 
Clin Immunol Immunopathol 1996 Sep;80(3 Pt 2):S40-5. 
44. 
Mqadmi A, Zheng X, Yazdanbakhsh K. CD4+CD25+ regulatory T cells control induction of 
autoimmune hemolytic anemia. Blood 2005 May 1;105(9):3746-8. 
45. 
Kuwana M, Kawakami Y, Ikeda Y. Suppression of autoreactive T-cell response to glycoprotein 
IIb/IIIa by blockade of CD40/CD154 interaction: implications for treatment of immune thrombocytopenic 
purpura. Blood 2003 Jan 15;101(2):621-3. 
46. 
Shevach EM. Regulatory T cells in autoimmmunity*. Annu Rev Immunol 2000;18:423-49. 


 
- 33 - 
47. 
Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in 
immunological tolerance to self and non-self. Nat Immunol 2005 Apr;6(4):345-52. 
48. 
Cataland SR, Ming J, Ferketich AK, et al. Cyclosporine and plasma exchange is superior to 
corticosteroids and plasma exchange as initial therapy of TTP. Blood 2005 November 15;106(11):359a. 
49. 
Jin M, Casper TC, Cataland SR, et al. Relationship between ADAMTS13 activity in clinical 
remission and the risk of TTP relapse. Br J Haematol 2008 Apr 7. 
50. 
Jin M, Cataland S, Bissell M, et al. A rapid test for the diagnosis of thrombotic thrombocytopenic 
purpura using surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF)-mass 
spectrometry. J Thromb Haemost 2006 Feb;4(2):333-8. 
51. 
Jin M, Casper TC, Cataland SR, et al. Relationship between ADAMTS13 activity in clinical 
remission and the risk of TTP relapse. Br J Haematol 2008 May;141(5):651-8. 
52. 
Kessler CM. An introduction to factor VIII inhibitors: the detection and quantitation. Am J Med 
1991 Nov 4;91(5A):1S-5S. 
53. 
Pinheiro JC, Bates DM. Mixed-effects models in S and S-PLUS. Springer, New York; 2000. 
54. 
Schall R. Estimation in Generalized Linear-Models with Random Effects. Biometrika 1991 
Dec;78(4):719-27. 
55. 
Bates DM, DebRoy S. Linear mixed models and penalized least squares. Journal of Multivariate 
Analysis 2004 Oct;91(1):1-17.