The Red Blood Cell Laboratory provides comprehensive services in red cell biology and hemoglobin disorders, and combines the Hemoglobinopathy laboratory at Children’s Hospital & Research Center at Oakland (CHRCO) and the Red Cell Research Laboratory at Children’s Hospital Oakland Research Institute (CHORI). Both laboratories are internationally recognized as a clinical resource for the diagnosis of hemoglobin and red cell disorders. These laboratories are an integral part of CHRCO's Comprehensive Thalassemia and Sickle Cell Centers and the Research Center for Sickle Cell Disease and Thalassemia at CHORI.
We provide both clinical diagnostics and research laboratory assays.
CLIA and CAP certified results of clinical diagnostics testing are released to referring health care professionals for use in diagnosis, counseling, and development of a treatment plan.
Translational studies in hemoglobinopathies thrive on a partnership between a research laboratory and clinical staff. Our laboratory provides comprehensive support in planning, execution and evaluation of translational studies. A large variety of research laboratory assays are performed under best laboratory practices: we will develop and implement new assays as needed for research studies.
The executive directors of the laboratories, Carolyn Hoppe, MD and Frans Kuypers, PhD have a long standing record in hemoglobinopathy and red cell research. The laboratory managers Mahin Azimi, BS and Sandra Larkin, MS have extensive experience in providing laboratory services for clinical, translational and basic studies.
In addition to the current routine measurements, we provide assays that are not on the list but have been established in the laboratory for specific clinical and translational studies.
One such recent example is shown as reported at the ASH meeting in December 2010 in Orlando Florida.
To support a translational study in sickle cell patients to evaluate the efficacy of a new compound to blunt the interaction between blood cells and endothelial cells, we developed in 2010 a panel of new assays. One of them measured the interaction between monocytes and platelets. The figure indicates the results of this assay performed with samples collected from patients one and 24 hours after receiving a dose of this compound. The results indicate that interaction between platelets and macrophages was reduced due to the presence of the compound. When the level of the compound decreased in the circulation, the double positive events increased again.
Phosphatidyl serine (PS) exposure has been shown to be increased in disease states such as sickle cell anemia and thalassemia.
A sample of erythrocytes is incubated with a fluorescently labeled dimer of the annexin V molecule, which binds specifically to PS in the presence of calcium. Any fluorescent events detected by flowcytometry are determined to expose PS and are counted as positive events.
Reticulocytes, young red blood cells from which the nucleus has been lost, contain residual organelles (ie, ribosomes and mitochondria), which differentiate reticulocytes from mature erythrocytes. An elevated reticulocyte count is expected in sickle cell disease, thalassemia, spherocytosis, glucose-6- phosphate dehydrogenase deﬁciency, immune hemolytic disease, or hypersplenism. However, normal or decreased numbers of reticulocytes are measured in individuals with impaired erythropoiesis, for example, immunologic or drug-induced red blood cell aplasia; metastatic carcinoma; decreased erythropoietin production; and iron, folate, or B12 deﬁciency anemias.
The reticulocyte count is a useful tool in clinical studies of new patient therapies. It can be used to evaluate a patient’s responsiveness to vitamin supplementation, follow the progress of bone marrow transplant, or assess the impact of chemotherapies on hematopoietic function.
The amount of reticulocytes as a percentage of total erythrocytes in human peripheral blood samples can be identified by RNA staining, or by the presence of a surface marker, both measured by flow cytometry, as well as by Advia 120 analysis. The membrane permeable Retic-COUNT (Fig. 2) reagent binds RNA within the reticulocytes, forming a ﬂuorescent nucleotide-reagent complex. The fluorescently labeled antibody to human CD71 (Fig. 1) binds to the transferrin receptor, a membrane glycoprotein involved in iron transport, on the surface of the youngest reticuloyctes. The Retic-COUNT reagent and the antibody reagent are for in vitro diagnostic use only. The normal range of reticulocytes for adults is 0.6 - 2.7% of total erythrocytes with a mean of 1.5%. The reticulocyte count assay results are for research use only.
Human Erythropoeitin (Epo), the principal regulating factor of erythropoiesis, is a glycoprotein produced primarily by the kidney and is regulated by changes in oxygen availability. Hypoxia causes an increase in the level of Epo in circulation and a resultant increase in RBC production. Abnormally high concentrations of serum Epo may be observed in various pathological states including anemia, polycythemia, renal neoplasms, benign tumors, polycystic kidney disease, renal cysts, and hydronephrosis.
The Epo ELISA is a standard sandwich enzyme immunoassay allowing for the quantitative determination of human Epo concentration in either fresh or frozen EDTA plasma or serum without prior sample purification. This ELISA is for research use only, not for diagnostic or therapeutic procedures. The standard curve spans the range of 2.5-200.0 mIU/ml and the minimum detectable level is 0.6 mIU/mL. If specimens generate values higher than the highest standard, specimens are diluted and repeated. The normal range of Epo concentration in human serum and plasma is 3 - 17 mIU/mL.
Oxygen Affinity, a red cell characteristic determined by both hemoglobin, and cytosolic composition is measured with the Hemox analyzer. The P50 is a conventional measure of the affinity of hemoglobin for oxygen. The oxyhemoglobin dissociation curve relates oxygen saturation (SO2) and partial pressure of oxygen in the blood (PO2). The PO2 at which the hemoglobin is 50% saturated, typically about 26-28 mmHg for a healthy person, is known as the P50. In the presence of disease or other conditions that change the hemoglobin's oxygen affinity and, consequently, shift the curve to the right or left, the P50 changes accordingly. An increased P50 indicates a rightward shift of the curve, which means that a larger partial pressure is necessary to maintain a 50% oxygen saturation. This indicates a decreased affinity. Conversely, a lower P50 indicates a leftward shift and a higher affinity.
Hemoglobin's affinity for oxygen increases as successive molecules of oxygen bind. More molecules bind as the PO2increases until the maximum amount that can be bound is reached. As this limit is approached, very little additional binding occurs and the curve levels out as the hemoglobin becomes saturated with oxygen. Hence the curve has a sigmoidal shape. At pressures above about 60 mmHg, the dissociation curve is relatively flat, which means that the oxygen content of the blood does not change significantly even with large increases in the PO2. Although binding of oxygen to hemoglobin continues to some extent for pressures below about 60 mmHg, as the PO2 decreases in this steep area of the curve, the oxygen is unloaded to peripheral tissue readily as the hemoglobin's affinity diminishes.
The effectiveness of hemoglobin-oxygen binding can be affected by several factors. The curve is shifted to the right by an increase in temperature, 2,3-diphosphoglycerate, PCO2, or a decrease in pH. The curve is shifted to the left by the opposite of these conditions, the presence of carbon monoxide, methemoglobinemia, and fetal hemoglobin. Typically, fetal arterial oxygen pressures are low, and hence the leftward shift enhances the placental uptake of oxygen.
The Hemox analyzer is an automatic system for the recording of blood oxygen equilibrium curves utilizing fresh whole blood based on dual wavelength spectrophotometry for the measurement of the optical properties of hemoglobin and an electrode for measuring the PO2 in mmHg. The resulting signals from both measuring systems are fed to the computer, which plots the resulting curve.
The production of 2,3 Diphosphoglycerate( 2,3 DPG) is an important function of the Embden-Meyerhoff pathway in the red cell. Together with ATP this phosphate easter is a regulator of the oxygen affinity of hemoglobin. A decrease in 2,3 DPG will shift the oxygen affinity curve to the left (lowers the P50) while an increase in 2,3 DPG will shift the oxygen affinity curve to the right (increase the P50). The measurement of 2,3, DPG levels is therefore important to properly evaluate the oxygen affinity of the red cell. Since the state of red cell metabolism will affect 2,3 DPG levels, fresh samples are required for measurement and special care needs to be taken in collection and shipment.
Human 2,3-Diphosphoglycerate (2,3-DPG) Assay
The 2,3-DPG Assay is a UV-detection for the determination of 2,3-DPG in blood samples in the range of 0.02–0.15 µmol in life science research applications. The 2,3-DPG in the sample is split by phosphoglycerate mutase to form phosphoglycerate, which is then detected at 340 nm.
This assay is for research use only and is not for use in diagnostics.
The normal range for adults is 4.8 ± 0.2 mM of 2,3-DPG/l erythrocytes and for children 5.3 ± 0.6 mM 2,3-DPG/l erythrocytes. The 2,3-DPG content within blood samples will change rapidly after collection. For this reason, the deproteinization procedure should be carried out immediately.
The deformability of erythrocytes (RBC) is measured in an ektacytometer, a laser-diffraction viscometer, as a continuous function of suspending medium osmolality. The ability of the RBC (diameter 6-8 µm) to pass through the microvasculature (openings of 2-3 µm) and splenic sinuses (openings of 0.5-1.0 µm) is referred to as deformability.
The resultant osmotic deformability profiles provide information on the viability of RBC, cellular water content, surface area, deformability of the cell, heterogeneity within these cellular properties, and several pathological conditions, which are characterized by typical shifts in this curve.
The osmotic deformability curve can be described by three parameters:
1) Omin is the osmolality at which the minimum deformability index (DI) is reached. This value is related to the surface area to volume ratio of the cell and has been found to equal the 50% hemolysis point in the classical osmotic deformability test.
2) Dimax is the maximum value of the deformability index, normally reached around 290 mosmol (the physiologic osmolality value). This indicates the maximum deformability of the cell under shear stress and is related to a number of factors, such as surface area, volume, internal viscosity, and mechanical properties of the cell membrane.
3) Ohyp is the osmolality at which the DI reaches half of its maximum value. This gives an indication of the position of the hypertonic part of the curve, which is related to the internal viscosity of the cell as well as mechanical properties of the membrane, such as how it will bend under force (stiffness)
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