anemia red blood cell disease
Conditions
Interventions
None listed
Sponsors
Universitair Medisch Centrum Utrecht
Eligibility
Age
2 Years to 99 Years
Inclusion criteria
Inclusion criteria: genetically confirmed diagnosis of pyruvate kinase deficiency
Exclusion criteria
Exclusion criteria: Patients may not have received blood transfusions within 3 months prior to blood collection.
Design outcomes
Primary
| Measure | Time frame |
|---|---|
| Main study parameter: PK antigen levels in patients with PK-deficiency. Red blood cell concentrates will be generated from 6 ml peripheral blood samples (EDTA) from PK-deficient patients and controls according to well-established methods. Subsequently, PK antigen levels will be determined by an ELISA method that has been recently developed in our laboratory (Van Wijk et al, 2009). Levels will be compared with normal control samples and correlated to the clinical phenotype and the genotype of patients. Van Wijk, R., Huizinga, E.G., Van Wesel, A.C.W., Van Oirschot, B.A., Hadders, M.A. & van Solinge, W.W. (2009) Fifteen novel mutations in PKLR associated with pyruvate kinase (PK) deficiency: structural implications of amino acid substitutions in PK. Hum Mutat, 30, 446-453. | — |
Secondary
| Measure | Time frame |
|---|---|
| Secondary study parameters: (1) Capability of human PK-deficient hematopoietic stem cells to differentiate into erythroid cells, and pro-apoptotic gene expression in PK-deficient erythroid cells. The peripheral blood mononuclear cells fraction will be obtained from 20 ml peripheral blood samples from PK-deficient patients and controls by density gradient centrifugation. Subsequently, these cells will be cultured in vitro in the presence of growth factors, including erythropoietin, for approximately 2 weeks. Erythroid colonies will be counted and characterized. Numbers and characteristics will be compared to normal control samples. After counting and characterization, erythroid colonies will be harvested and total RNA will be isolated. This RNA will be used to study expression of pro-apoptotic genes like, for example, BAD, BNIP3, and BNIP3l by quantitative RT-PCR methods. Gene expression levels will be calculated relative to normal control expression levels. (2) PK-deficient red blood cell characteristics (red blood cell counts), iron status, ane erythropoietin levels. In order to interpret results and to classify PK-deficient patients according to their fenotype, general red blood cell parameters (e.g. hemoglobin, reticulocyte counts), the patient's iron status as well as erythropoietin levels should be taken into account. For this, 6 ml lithium-heparine (erythropoietin levels) and 3 ml EDTA peripheral blood samples will be collected from PK-deficient patients. Laboratory tests will be performed by the Central Diagnostic Laboratory of the Department of Clinical Chemistry and Haematology. Results will be compared with the reference values of the laboratory. | — |
Countries
Netherlands
Outcome results
None listed