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This site is designed to provide information about, and aid recruitment into, an ongoing research study evaluating peripheral blood samples from patients with hemolytic anemia.
What is the rationale for this study?
Who is eligible for participation in this study?
Can transfusion dependent patients participate?
What sample is required for study, and how are patients enrolled?
How are patient samples analyzed?
What type of data is communicated back to the referring physician/family?
What is the rationale for this study?
Hereditary hemolytic anemia, comprising both enzyme deficiencies and red cell membrane protein defects are a heterogeneous group of disorders that present a continuing diagnostic challenge; testing remains time consuming and highly specialized. The diagnostic challenge in red cell enzyme disorders is exemplified by a failure (even in the most experienced laboratories) to establish a diagnosis in more than 3/4 of cases examined (according to data compiled by S. Miwa in Japan, and E. Beutler in the United States). The diagnostic challenge in presumed membrane protein defects is different—most cases can eventually be related to specific mutations, but the procedures involved are highly specialized, costly and time consuming.
To address these problems with diagnosis, NIDDK/NIH is supporting research aimed at identifying additional causes of hereditary hemolytic anemia. Two novel approaches to diagnosis will be applied to blood samples from patients with hemolytic anemia:
1) Comparison of the red cell proteome of patients versus normal controls using 2D difference gel analysis (DIGE)
A brief description and representative data using this methodology can be viewed on the ‘for patients’ page by clicking here.
2) Measurement of damage due to reactive oxygen species in patient samples versus controls.
Samples will undergo standard enzyme studies in parallel so that we may compare proteomics against current methods for establishing a diagnosis. Because proteomics has the potential to identify novel proteins/genes involved in the pathogenesis of these disorders, we expect future effort to be devoted to characterization of differentially expressed proteins (and their corresponding genes) identified through these analyses.
Who is eligible for participation in this study?
Patients with chronic, nonspherocytic hemolytic anemia are the target population for this study. As we are focused upon potential hereditary cases, presentation at a young age and/or a positive family history of anemia is desired. Prior workup to exclude hemoglobinopathy, immune mediated hemolysis and the most common enzyme deficiencies (G6PD and PK) is requested.
Can transfusion dependent patients participate?
Transfusion dependent patients can participate provided a sample is obtained just prior to transfusion. In such cases, our analysis relies upon purification and characterization of reticulocytes, and may not include all of the analyses listed below.
What sample is required for study, and how are patients enrolled?
We request 7-14 cc of peripheral blood collected in purple top (EDTA) tubes. Samples are placed on wet ice and shipped via overnight courier to our lab in at the Scripps Research Institute in La Jolla, California for analysis. Patients are enrolled in the study by the referring physician using the Scripps Research Institute IRB approved consent form. As many of our patients are children, we also encourage enrollment of both parents to provide control samples. These control samples are particularly helpful in proteomic comparisons—allowing for discrimination of polymorphic variants from potentially causal variants.
How are patient samples analyzed?
Fresh samples are assessed for selected red cell enzymatic activities (G6PD, Pyruvate Kinase, Triose Phosphate Isomerase, Glutathione Peroxidase, Hexokinase, Adenylate Kinase, Phosphoglycerate Kinase, Glucose Phosphate Isomerase), reduced glutathione and unstable hemoglobins. Reticulocytes are measured, and purified reticulocytes are stored for subsequent evaluation. Red cells are incubated in the presence of reactive oxygen (ROS) sensitive dyes to measure basal and provoked ROS production. Packed red cells are prepared for subsequent proteomic analysis—reserved for those cases where no known cause for hemolytic anemia is identified using the above screening tests.
What type of data is communicated back to the referring physician/family?
Any abnormalities in the screening tests are communicated back to the referring physician for further evaluation. It is important to point out that our screening tests are not a substitute for evaluation in a clinical laboratory—and our results should be considered ‘research only.’ Results from proteomic experiments will be communicated back to the referring physician/family in those cases where a potentially causal protein variation is identified. In such cases, evaluation of the corresponding region of genomic DNA will be performed in an attempt to confirm the protein results. The results of this evaluation will also be provided to the referring physician. The time frame for results of screening assays is 2-3 weeks, while proteomic data requires several months.
For more information on this research study or information on enrolling patients, please contact Dr. Jeff Friedman at the Scripps Research Institute Friedman@scripps.edu.
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