Rituximab for treating inhibitors in children with hemophilia

  • Protocol
  • Intervention


  • Yi Liu,

    1. West China Second University Hospital, Sichuan University, Department of Pharmacy, Evidence-based Pharmacy Center, Chengdu, Sichuan, China
    2. Sichuan University, Key Laboratory of Birth Defects and Related Diseases of Women and Children, Chengdu, China
    3. Sichuan University, West China Second University Hospital, Chengdu, China
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  • Lingli Zhang,

    Corresponding author
    1. West China Second University Hospital, Sichuan University, Department of Pharmacy, Evidence-based Pharmacy Center, Chengdu, Sichuan, China
    2. Sichuan University, Key Laboratory of Birth Defects and Related Diseases of Women and Children, Chengdu, China
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  • Cristina Santoro,

    1. Sapienza University of Rome, Department of Hematology and Cellular Biotechnology, Rome, Italy
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  • Jie Song,

    1. West China Second University Hospital, Sichuan University, Department of Pharmacy, Evidence-based Pharmacy Center, Chengdu, Sichuan, China
    2. Sichuan University, Key Laboratory of Birth Defects and Related Diseases of Women and Children, Chengdu, China
    3. Sichuan University, West China Second University Hospital, Chengdu, China
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  • Armando Rodriguez,

    1. Association for the Promotion of Multimedia Education, Zagreb, Croatia
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  • Li Wang

    1. West China Hospital, Sichuan University, Chinese Cochrane Centre, Chengdu, Sichuan, China
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This is the protocol for a review and there is no abstract. The objectives are as follows:

To assess the efficacy and safety of rituximab in the treatment of clotting factor inhibitors in children with hemophilia.


Description of the condition

Hemophilia is a rare, inherited, X-linked, recessive disorder in which the blood does not clot normally (NHLBI 2011). Treatment consists of administering concentrated FVIII (for hemophilia A) or FIX (for hemophilia B) as required (when bleeding occurs), or prophylactically to prevent bleeding (Iorio 2011 Coppola 2012). Inhibitors can occur when the body's immune system stops accepting clotting factor concentrates as a normal part of the blood (WFH 2013). These inhibitors reduce the efficacy of, or even eliminate, clotting factor concentrates. There are several known risk factors for the occurrence of inhibitors; either genetic risk factors (nature of gene defect, ethnicity) or non-genetic risk factors (intensive factor exposure at the time of surgery, and prophylactic or on-demand treatment regimens) (Iorio 2010a). The source of FVIII used for replacement therapy may also have an effect on inhibitor development. Studies have shown that for previously untreated patients (PUPs) with severe hemophilia A, high-dose (HD) intensive FVIII treatment increases the risk for inhibitor development, but low-dose (LD) prophylactic treatment decreases it, especially in patients with low-inhibitor-risk FVIII genotype mutations (Gouw 2007a; Gouw 2013a). There is no significant difference in the risk of inhibitor development between recombinant or plasma-derived therapy (Gouw 2007b; Gouw 2013b). 

Blood screening tests can show the presence of a bleeding disorder. The activated partial thromboplastin time (APTT) test often suggests the presence of inhibitors, either when the sample is non-coagulable or has a prolonged clotting time, which is not corrected (either at all or partially) by mixing with normal plasma. The Nijmegen assay is performed to confirm diagnosis, which can determine the titer of inhibitors (Verbruggen 1995). The amount of inhibitors in the blood is measured in Bethesda units (BU), where a level of more than five BU is referred to as 'high titer' and less than five BU as 'low titer' (WFH 2012a). Inhibitors usually occur between the first 10 to 20 treatments and therefore most often develop in childhood. Approximately 30% of children with hemophilia A (Gouw 2013b), and 1% to 6% of individuals with hemophilia B (WFH 2012b), develop inhibitors against the clotting factor. Sometimes the bleeding is spontaneous; surgical operations and strenuous exercise can be precipitating factors to acute bleeding.

Hemophilia patients with inhibitors commonly receive bypassing agents for acute bleeding episodes and immune tolerance induction (ITI) to eliminate inhibitors. Bypassing agents, such as recombinant FVIIa concentrate (rFVIIa) or activated prothrombin complex concentrate (aPCC), are insensitive to inhibitors (Iorio 2010b). As an alternative, and where resources are not a limitation, bypassing agents can be used prophylactically (Leissinger 2011). The standard treatment, ITI, is the regular infusion of FVIII in an attempt to achieve immunogenic acceptance (immunologic tolerance). Success is no different between using a HD or LD of therapy, although HD therapy can achieve success more rapidly (Hay 2012). Patients with hemophilia B and high-titer FIX inhibitors may also be treated with ITI, although this approach is successful far less frequently than in patients with hemophilia A (DiMichele 2007). However, ITI is expensive and not always effective for everyone; it requires specialized medical expertise over a long period of time.

Patients with inhibitors continue to have significantly more issues with morbidity and mortality, with treatment costs being considerable. The ultimate eradication of inhibitors remains the most challenging treatment issue in this group of patients.

Description of the intervention

Many immunomodulatory approaches have been studied in the search for more effective techniques to eliminate clotting factor inhibitors. The chimeric monoclonal antibody, rituximab, shows particular promise. In 1997, the USA's Food and Drug Administration (FDA) approved rituximab, also known as MabThera® (F. Hoffmann-La Roche Ltd., Pharmaceuticals Division, Basel, Switzerland) and Rituxan® (IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc, San Francisco, CA), for use in adult CD20+ B-cell lymphomas (Kavcic 2013). So far, this treatment is licensed for the following indications in adults: non-Hodgkin's lymphoma (NHL); chronic lymphocytic leukemia (CLL); rheumatoid arthritis (RA); granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis); and microscopic polyangiitis (MPA) (FDA 2013). In pediatric hematology-oncology departments, rituximab is widely used off-label (Yadav 2012) to treat such diseases as chronic immune thrombocytopenia (ITP) (Grace 2012), systemic lupus erythematosus (SLE) (Nwobi 2008) and autoimmune hemolytic anemia (Kuzmanovic 2012). Although given its off-label use, studies in the pediatric population are limited. The standard single dose of rituximab for inhibitors is 375 mg/m2, administered intravenously on a weekly schedule for four weeks. Rituximab currently is available in a 10 mg/mL concentrate of either 10 mL (100 mg, average wholesale price $568) or 50 mL (500 mg average wholesale price $2840) (Pescovitz 2006). It is an expensive drug and not covered by many insurance plans or by government funding.

How the intervention might work

Rituximab is a chimeric mouse-human monoclonal immunoglobulin G1 antibody against the CD20 antigen on the surface of B lymphocytes (Borker 2011). The binding of rituximab to CD20, which is a B-cell differentiation maker, may cause B-cell death that produces inhibitors in three ways, including complement-dependent cytotoxicity (CDC), stimulation of apoptosis, or antibody-dependent cell-mediated cytotoxicity (ADCC) (Selewski 2010). Rituximab targets the CD20+ B-cells, with a rapid and sustained elimination. Generally, rituximab is used as second-line treatment for inhibitors, if a patient is resistant to ITI. It is hypothesized that rituximab can reduce inhibitor titre to facilitate ITI in resistant inhibitor cases. A national cohort in the UK has showed the efficacy of this treatment (Collins 2009). Rituximab has been used for treating people with hemophilia with inhibitors with a response rate of up to 63% (Borker 2011).

Why it is important to do this review

Two non-Cochrane systematic reviews have examined the literature on rituximab for treating children with hemophilia who express clotting factor inhibitors. One review, published in 2008, was based on studies including only patients with congenital hemophilia, and it did not report conclusions specifically about children (Franchini 2008). Another review, published in 2007, included eight children with hemophilia (Giulino 2007). These two reviews included only case reports and case series involving a relatively small number of patients, making the reviews potentially biased and imprecise. The two reviews were unable to draw definitive conclusions or make recommendations about using rituximab to manage hemophilia in children with clotting factor inhibitors.

Although rituximab has become widely available as a therapy option, its high price and the lack of definitive evidence about its efficacy and safety remain barriers to its acceptance by patients and physicians. To guide both groups as they decide on the most appropriate hemophilia treatment, we will undertake a Cochrane systematic review to evaluate the efficacy and safety of rituximab to eradicate inhibitors complicating congential hemophilia treatment in children.


To assess the efficacy and safety of rituximab in the treatment of clotting factor inhibitors in children with hemophilia.


Criteria for considering studies for this review

Types of studies

Randomized and quasi-randomized controlled trials (RCTs; quasi-RCTs). Controlled clinical trials will be considered if no RCTs or quasi-RCTs are found.

Types of participants

Children up to (not including) 18 years of age with confirmed congenital hemophilia A or B and inhibitors.

Acceptable methods to diagnose inhibitor cases will be as follows.

  1. A positive result of the Nijmegen assay performed for monitoring the titer of inhibitors in asymptomatic patients (Verbruggen 1995).

  2. A positive result of the Nijmegen assay performed to confirm the presence of blood clotting inhibitors suspected on the basis of the following signs and symptoms (WFH 2012c):

    1. standard doses of concentrated clotting factor do not adequately control bleeding;

    2. standard treatment with concentrated clotting factor gradually becomes less effective;

    3. bleeding becomes gradually more difficult to control;

    4. bleeding occurs spontaneously in atypical sites (eg mucocutaneous).

Types of interventions

Intervention group: intravenous rituximab, irrespective of dose or of duration and interval of administration.

Control group: placebo, no treatment, or intervention not involving rituximab.

Adjunct therapy, if recommended or prescribed, should be given to both groups. Different doses of rituximab treatment in different groups will be compared.

Types of outcome measures

Primary outcomes
  1. Remission or no response (Hay 2012)

    1. complete remission: negative inhibitor titer, FVIII recovery > 66% of expected, and FVIII recovery > 6 hours

    2. partial remission: negative inhibitor titer, but persistently abnormal recovery or half-life; responding clinically to FVIII replacement without an anamnestic increase in inhibitor titer

    3. failure: failure of the inhibitor to decline by 20% over any six-month period; or failure to achieve tolerance or partial response; or withdrawal from the study for any reason before tolerance was achieved

    4. relapse: inhibitor recurrence during the 12-month follow-up period on prophylaxis after tolerance was achieved, as evidenced by recurrent positive Bethesda titer or a decline in FVIII recovery or half-life below study limits.

Secondary outcomes
  1. Months to complete remission (time-to-event data)

  2. Duration of remission

  3. Relapse rate

  4. Cost of care

  5. Quality of life (as reported within trials or specifically using validated questionnaires, etc)

  6. Mortality

  7. Adverse drug reactions

Search methods for identification of studies

Electronic searches

We will identify relevant studies from the Cystic Fibrosis and Genetic Disorders Group's Coagulopathies Trials Register using the terms: haemophilia* AND rituximab.

The Coagulopathies Register is compiled from electronic searches of the Cochrane Central Register of Controlled Trials (CENTRAL) (updated each new issue of The Cochrane Library), quarterly searches of MEDLINE and prospective hand-searching of one specialized journal, Haemophilia. Unpublished work is identified by searching the abstract books of five major conferences: the European Haematology Association conference; the American Society of Hematology conference; the British Society for Haematology Annual Scientific Meeting; the International Society of Haemostasis and Thrombosis Congresses; and the International Congresses of World Federation of Haemophilia. For full details of all searching activities for the register, please see the relevant section of the Cochrane Cystic Fibrosis and Genetic Disorders Group Module.

Bibliographic databases: we will also retrieve articles from the following databases: PubMed, Embase, the Chinese National Knowledge Infrastructure (CNKI) database, the Chinese Biomedical Literature Database (CBM), VIP, WANFANG and LILACS. We searched all databases from their earliest records to September 2013. Detailed search strategies are given in an appendix (Appendix 1; Appendix 2).

Searching other resources

  1. Researchers: we will contact the major researchers in this field to gather information on unpublished or ongoing trials.

  2. OpenGrey or Clinicaltrials.gov.

  3. Organizations: we will analyze the web sites of the following organizations to identify grey literature: the Canadian Hemophilia Society; the National Hemophilia Foundation; the National Heart, Lung and Blood Institute (USA); the Haemophilia Alliance.

  4. Conferences: the European Haematology Association (EHA) conference; the American Society of Haematology (ASH) conference; the British Society for Haematology (BSH) Annual Scientific Meeting; the Congress of the International Society of Thrombosis and Haemostasis (ISTH); and the Congress of the World Federation of Haemophilia (WFH).

  5. Pharmaceutical companies: we will contact the pharmaceutical companies: F. Hoffmann-La Roche Inc.; IDEC Pharmaceuticals; and Genentech, Inc. for information on unpublished or ongoing trials.

  6. Reference lists: we will manually search the reference lists of relevant reviews, systematic reviews and original research studies.

Data collection and analysis

Selection of studies

Two authors will independently scan the titles and abstracts of all reports identified from the literature searches. The complete articles will be obtained if we need more information beyond what is in the title and abstract. Two authors will independently apply the inclusion criteria to identified articles in order to determine whether we should include these in the review or not. We will resolve disagreements by discussion among co-authors and, when necessary, with the Cochrane Cystic Fibrosis and Genetic Disorder Group. We will use reference management software to merge search results and remove duplicate records. We will also scrutinize each study to ensure that no trial is reported in multiple publications. We will document reasons for excluding studies.

Data extraction and management

Two authors will independently extract data using pre-designed forms. These extracted data will include characteristics of the participants, interventions and outcomes. The authors will test and optimize the data extraction form before extraction after a preliminary test. If data for a study are incomplete, the review authors will contact the primary study author for further information and clarification. The authors aim to detect and resolve any discrepancies by discussion and cross-checking.

If any included studies involve children and adults we will attempt to extract data separately for children. If any studies examine hemophilia A or B with inhibitors in addition to other hematological diseases, we will include these and attempt to extract the relevant data.

Assessment of risk of bias in included studies

Two authors will independently assess the risk of bias of included studies using a standardized assessment form. The authors will assess bias in RCTs across the following six domains according to the domain-based evaluation recommended in the Cochrane Handbook for Systematic reviews of Interventions (Higgins 2011a): sequence generation; allocation concealment; blinding of participants and personnel; blinding to outcome assessment; incomplete outcome data; and selective outcome reporting. If we are unable to obtain important information from the published studies, we will contact the primary author of the relevant study. We will aim to resolve any disagreements by discussion.

Measures of treatment effect

To measure treatment effects, we will use the rate ratio (RR) or odds ratio (OR) for dichotomous data; we will also use the RR to measure time-to event data. We will use the mean difference (MD) for measuring continuous data. If studies report the same outcome on different scales which cannot be converted to the same unit, we will use the standardized mean difference (SMD). We will report all of these measurements with their corresponding 95% confidence intervals (CI).

Unit of analysis issues

We will include cluster-randomised trials and cross-over trials along with individual-randomised trials. In this systematic review, we will treat each group or cluster in cluster-randomised trials as the unit of analysis; we will use the intracluster correlation coefficient (ICC) to estimate the relative variability within and between clusters, according to the relevant section of the Cochrane Handbook of Systematic Reviews of Interventions (Higgins 2011b). For cross-over trials with binary outcomes, we will adopt the Becker-Balagtas approach to combine cross-over trials with parallel trials for meta-analysis (Stedman 2011). For cross-over trials with continuous outcomes, each individual will act as the unit of analysis and we will conduct approximate paired analyses (Higgins 2011b).

Dealing with missing data

If data on individuals are missing, such as when randomized participants are excluded from the analysis, we will conduct intention-to-treat analyses. We will follow the principal options and adopt four general recommendations for dealing with missing data, as described in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011b). These are:

  1. whenever possible, we will contact the original investigators to request missing data;

  2. we will make explicit the assumptions of any methods used to cope with missing data: for example, that the data are assumed missing at random, or that missing values were assumed to have a particular value such as a poor outcome;

  3. we will perform sensitivity analyses to assess how sensitive results are to reasonable changes in the assumptions that are made;

  4. we will address the potential impact of missing data on the findings of the review in the discussion section.

If necessary, and possible, the authors plan to impute any missing standard deviations (SDs).

Assessment of heterogeneity

The authors will identify any statistical heterogeneity among the included studies based on the chi-squared test ( χ2 or Chi2) included in the forest plots. A P value of ≤ 0.05 will be taken as evidence of heterogeneity in treatment effects, and the I2 statistic will be used to quantify the heterogeneity. We will interpret the level of heterogeneity according to the Cochrane Handbook for Systematic reviews of Interventions (Higgins 2011a): 0% to 40%, may not be important; 30% to 60%, may represent moderate heterogeneity; 50% to 90%, may represent substantial heterogeneity; 75% to 100%, represents considerable heterogeneity. We will generally consider heterogeneity to be acceptable if the I2 statistic is ≤ 50%. The authors will also refer to the forest plots to see if the CIs overlap in order to further identify any possible heterogeneity.

Assessment of reporting biases

If we include 10 or more studies, the authors will use a funnel plot to detect small-study effects, including publication bias. They will use a linear regression approach proposed by Egger to test for funnel plot asymmetry (Egger 1997).

Data synthesis

The authors will use Review Manager 5.2 to analyze data (RevMan 2011). If the studies show homogeneity, we will carry out a meta-analysis of pooled research outcomes using a fixed-effects model. If the studies show heterogeneity, we will use a random-effects model or describe the results narratively. If we are unable to undertake any meta-analyses, we will summarize the study results in tables.We will describe the time to response and response duration with medians, minimum and maximum values. If we are able to include sufficient data, we will group outcome data for analysis at six months, one year and annually thereafter. However, if outcome data were recorded at other time periods, then we will consider examining these as well.

Subgroup analysis and investigation of heterogeneity

If ten or more studies are included, and if we identify heterogeneity (I2 ≤ 50%), we plan to investigate this by performing the following sub-group analyses:

  1. hemophilia groups: hemophilia A; hemophilia B;

  2. hemophilia severity groups: moderate; severe;

  3. treatment groups: rituximab alone; rituximab plus other treatment.

Sensitivity analysis

If the authors are able to include at least 10 trials in the review, we will perform a sensitivity analysis to determine whether the conclusions are robust to decisions made during the review process. We therefore plan to perform sensitivity analyses by re-analyzing the data statistically, such as using a fixed-effects model first and then a random-effects model, and vice versa.


Many thanks to all authors' affiliated Institutions and Organizations, and thank the co-ordinating editors, referees and editors in Cochrane Cystic Fibrosis and Genetic Disorders Group for their comments and encouragement.


Appendix 1. Detailed search strategies

No. PubMed Embase LILACS
#2"Hemophilia A/drug therapy"[Mesh]'hemophilia'/expHemophilia/Haemophilia
#3"Hemophilia B/drug therapy"[Mesh]'rituximab'/expRitux$
#4"rituximab" [Supplementary Concept]Ritux*rituximab
#5Ritux*'mabthera'/exp#1 OR #2
#6Mabtheramabthera#3 OR #4
#7"Antibodies, Monoclonal, Murine-Derived/therapeutic use"[Mesh]Antibodies#5 AND #6
#10#8 AND #9#1 OR #2 
#11 #3-#6/OR 
#12 #7-#9/AND 
#13 #11 OR #12 
#14 #10 AND #13 

Appendix 2. Database Weblink




Appendix 3. Definition

  • Complement-dependent cytotoxicity (CDC): Binding of the antibody recruits complement proteins, which punch holes in the cell membrane, flooding the cell and leading to celllysis

  • Apoptosis: Binding of the antibody signals the cell to self-destruct

  • Antibody-dependent cell-mediated cytotoxicity (ADCC): Natural killer cells, T cells, and macrophages are involved in recognizing and killing antibody-labeled target cells, leading to celllysis

Contributions of authors

 Roles and responsibilities
Task Who will undertake the task?
Protocol stage: draft the protocolYi Liu; Cristina Santoro; Armando Rodriguez
Review stage: select which trials to include (2 + 1 arbiter)Yi Liu; Jie Song; Lingli Zhang
Review stage: extract data from trials (2 people)Yi Liu; Jie Song
Review stage: enter data into RevManYi Liu; Jie Song
Review stage: carry out the analysisLingli Zhang; Li Wang
Review stage: interpret the analysisLi Wang; Cristina Santoro
Review stage: draft the final reviewYi Liu; Cristina Santoro; Armando Rodriguez
Update stage: update the reviewYi Liu; Lingli Zhang

Declarations of interest

None Known.

Sources of support

Internal sources

  • Sichuan University, China.

    Free Databases

  • West China Second University Hospital,Sichuan University, China.

    Free Database

External sources

  • Natural Science Foundation of China: Evidence based establishment of evaluation index system for pediatric rational drug use in China (No. 81373381), China.