Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics.

BACKGROUND: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. RESULTS: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. CONCLUSION: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia.

Spherocytic red cells with reduced membrane surface area are a feature of hereditary spherocytosis (HS) and some forms of autoimmune hemolytic anemia (AIHA). It is generally assumed that membrane loss in spherocytic red cells occurs during their sojourn in circulation. The structural basis for membrane loss in HS is improper assembly of membrane proteins, whereas in AIHA it is due to partial phagocytosis of circulating red cells by macrophages. A hypothesis was formed that these different mechanisms should lead to temporal differences in surface area loss during red cell genesis and during sojourn in circulation in these 2 spherocytic syndromes. It was proposed that cell surface loss could begin at the reticulocyte stage in HS, whereas surface area loss in AIHA involves only circulating mature red cells. The validity of this hypothesis was established by documenting differences in cellular features of reticulocytes in HS and AIHA. Using a novel technique to quantitate cell surface area, the decreased membrane surface area of both reticulocytes and mature red cells in HS compared with normal cells was documented. In contrast, in AIHA only mature red cells but not reticulocytes exhibited decreased membrane surface area. These data imply that surface area loss in HS, but not in AIHA, is already present at the circulating reticulocyte stage. These findings imply that loss of cell surface area is an early event during genesis of HS red cells and challenge the existing concepts that surface area loss in HS occurs predominantly during the sojourn of mature red cells in circulation.

Identification and characterization of a newly recognized population of high-Na+, low-K+, low-density sickle and normal red cells.

We describe a population of sickle cell anemia red cells (SS RBCs) ( approximately 4%) and a smaller fraction of normal RBCs (<0.03%) that fail to dehydrate when permeabilized to K(+) with either valinomycin or elevated internal Ca(2+). The nonshrinking, valinomycin-resistant (val-res) fractions, first detected by flow cytometry of density-fractionated SS RBCs, constituted up to 60% of the lightest, reticulocyte-rich (R1) cell fraction, and progressively smaller portions of the slightly denser R2 cells and discocytes. R1 val-res RBCs had a mean cell hemoglobin concentration of approximately 21 g of Hb per dl, and many had an elongated shape like "irreversibly sickled cells," suggesting a dense SS cell origin. Of three possible explanations for val-res cells, failure of valinomycin to K(+)-permeabilize the cells, low co-ion permeability, or reduced driving K(+) gradient, the latter proved responsible: Both SS and normal val-res RBCs were consistently high-Na(+) and low-K(+), even when processed entirely in Na-free media. Ca(2+) + A23187-induced K(+)-permeabilization of SS R1 fractions revealed a similar fraction of cal-res cells, whose (86)Rb uptake showed both high Na/K pump and leak fluxes. val-res/cal-res RBCs might represent either a distinct erythroid genealogy, or an "end-stage" of normal and SS RBCs. This paper focuses on the discovery, basic characterization, and exclusion of artifactual origin of this RBC fraction. Many future studies will be needed to clarify their mechanism of generation and full pathophysiological significance.

Genetic correction of sickle cell disease: insights using transgenic mouse models.

Sickle cell disease is a hereditary disorder characterized by erythrocyte deformity due to hemoglobin polymerization. We assessed in vivo the potential curative threshold of fetal hemoglobin in the SAD transgenic mouse model of sickle cell disease using mating with mice expressing the human fetal Agamma-globin gene. With increasing levels of HbF, AgammaSAD mice showed considerable improvement in all hematologic parameters, morphopathologic features and life span/survival. We established the direct therapeutic effect of fetal hemoglobin on sickle cell disease and demonstrated correction by increasing fetal hemoglobin to about 9-16% in this mouse model. This in vivo study emphasizes the potential of the SAD mouse models for quantitative analysis of gene therapy approaches.

Reticulocyte hemoglobin: an integrated parameter for evaluation of erythropoietic activity.

Traditional reticulocyte counts provide only a partial estimate of the erythropoietic bone marrow activity and do not account for qualitative variations of reticulocyte cellular indexes and hemoglobin content in particular. We have studied a new integrated parameter, reticulocyte hemoglobin (retHb), that quantifies in grams per liter the hemoglobin contained in the circulating reticulocyte compartment and is obtained by multiplying the absolute reticulocyte count and the reticulocyte cell hemoglobin content. In 50 normal control subjects, retHb values were 1.76 +/- 0.59 g/L. The retHb values were lower in patients double heterozygous for HbS and HbC (SC disease) (3.33 +/- 1.52 g/L, n = 13) compared with homozygous HbS disease (SS) with concomitant alpha-thalassemia (5.27 +/- 1.51 g/L and 5.48 +/- 1.06 g/L for 12 patients with 3 alpha-genes and 3 patients with 2 alpha-genes, respectively) and to SS disease with no alpha-thalassemia (6.47 +/- 3.05, n = 20). The hemoglobin contained in the red blood cell pool (rbcHb) also can be calculated by subtracting retHb from the total hemoglobin. The ratio between the two pools (rbcHb/retHb, normal value 76.6 +/- 21.9, n = 50) provides a rough estimate of red blood cell survival. It was 9.8 +/- 4.1 in SS disease, 16.2 +/- 10.1 and 14.7 +/- 5.0 in SS disease with 3 and 2 normal alpha-genes, respectively, and 36.6 +/- 17.8 in SC disease with no alpha-thalassemia. We also studied retHb in patients receiving hydroxyurea therapy for SS disease, intravenous or oral iron for iron deficiency, or recombinant human erythropoietin (r-HuEPO) therapy. All these conditions are characterized by changes in reticulocyte counts and marked variations in reticulocyte cellular hemoglobin contents, which can be integrated into the retHb parameter. Measurement of retHb and the rbcHb/retHb ratio may provide an estimate of the reduction in red blood cell survival and the severity of hemolysis in various anemias and allow more precise monitoring of the response to hydroxyurea, iron, r-HuEPO, or other therapies.

Generation of normal human red cell volume, hemoglobin content, and membrane area distributions by "birth" or regulation?

Using flow cytometry and osmotic lysis measurements, we document here the means and coefficients of variation of the following red cell (RBC) properties: hemoglobin (Hb) content, volume, Hb concentration, and relative lytic tonicity distributions in populations of normal human RBCs, before and after density fractionation. The distributions showed a pattern characterized by much larger coefficients of variation of the Hb content and volume distributions than of the Hb concentration and relative lytic tonicity distributions. From analysis of the factors that determine those RBC properties, the patterns were interpreted as reflecting previously unrecognized statistical proportionalities between cell osmolyte content, Hb content, and membrane area. The possible origin of these statistical links was analyzed by considering alternative models with and without the participation of regulatory processes during cell maturation. A model was shown to be feasible in which mature RBC variability with proportional volume, area, and Hb content arises solely from cell size variability at the last erythroid cell division.

Sickle cell disease of transgenic SAD mice.

Erythrocyte sickling on deoxygenation in vitro occurs in transgenic SAD mice, hemizygous for a modified human sickle hemoglobin, HbSAD [alpha 2 beta 2S(beta 6val)Antilles (beta 23 lle)D- Punjab (beta 121Gln)] (SAD-1, 19% HbSAD; beta-thal/SAD-1, 26% HbSAD). The present study examines the cellular defects in vivo and pathologic changes observed in SAD-1 mice at atmospheric oxygenation as well as the effect of acute hypoxia. The transgenic mice showed generalized congestion and microvascular occlusions, occasionally with thrombosis and infarctions of lung, kidneys, penis, and myocardium. The most prevalent chronic organ lesions were congestive splenomegaly (83% of animals) and renal glomerulopathy, which affected 75% of animals by 10 months of age. Further, SAD mice have a mean lifespan that was reduced by 40% when compared with nontransgenic littermates. Premature death of SAD mice was associated with acute vasoocclusive events or severe renal disease. SAD mice developed lethal vasoocclusive processes when exposed to reduced pO2 conditions, whereas control mice survived normally. The sensitivity to hypoxia appears to depend on the cellular level of HbSAD, because death occurred at pO2 of 42 mmHg for SAD mice and 49 mmHg for beta-thal/SAD. Administration of an antisickling agent that increases oxygen affinity (BW12C79) protected SAD and beta-thal/SAD mice from the lethal hypoxic stress. In conclusion, the transgenic SAD and beta-thal/SAD mice developed a pathophysiology that strongly resembles human sickle cell disease. Moreover, this animal model allows studies on the effect of antisickling agents.

Improved isolation of normal human reticulocytes via exploitation of chloride-dependent potassium transport.

Studies on normal human reticulocytes have been limited by a lack of methods for effective reticulocyte enrichment. This study shows a convenient new approach for selective enrichment of reticulocytes from normal blood samples. We have developed a modified arabinogalactan density gradient that contains high potassium levels, approximating the internal cation composition of red blood cells (RBC). The low-density populations from this gradient are enriched in reticulocytes, and the highly selected lowest density fraction shows a much higher reticulocyte enrichment than that obtained with high sodium chloride arabinogalactan density gradients, or other previously reported density gradient methods. We found that this improved isolation is caused by suppression of potassium loss and reticulocyte dehydration via chloride (KCI) cotransport. When the low-density fraction of RBC from a high-potassium gradient was subsequently incubated in high sodium chloride medium and reseparated on a sodium chloride density gradient, the reticulocytes dehydrated and were recovered in high-density fractions. The highest-density fractions from this secondary gradient yield 95% to 99% reticulocytes. We anticipate that this method will benefit investigators who require reticulocyte enriched populations for a wide variety of applications.

Prehemolytic effects of hydrogen peroxide and t-butylhydroperoxide on selected red cell properties.

To provide further understanding of how oxidative damage affects red cell membrane function, the effects of low levels of two different types of oxidants on selected red cell properties have been studied. Hydrogen peroxide (H2O2), an example of a water soluble oxidant, and t-butylhydroperoxide (tBHP), a hydrophobic hydroperoxide, were compared with respect to their effects on membrane permeability, membrane mechanical properties and binding of autologous serum antibodies to the cell surface. Whereas H2O2 treatment resulted in a dose-dependent increase in membrane permeability to potassium that was evident after one hour of oxidant exposure, cells treated with tBHP at doses up to 5 mumol/ml cells showed no immediate change in cation permeability. H2O2 also caused a marked decrease in membrane deformability, whereas tBHP-treated cells showed minimal loss of deformability. However, tBHP treatment did result in a dose-dependent increase in the susceptibility of the membrane to fragmentation under high shear stress. With exclusion of treated samples that bound excess rabbit anti-spectrin antibody, indicating exposure of intracellular components, neither agent promoted the binding of autologous serum antibody in amounts comparable to that found in vivo on high density or some pathologic red cells. Taken together, the results suggest that tBHP and H2O2 cause damage to human red cells by distinct oxidative mechanisms which do not lead directly to substantive generation of binding sites for autologous serum antibodies.

Specificity of autologous antibody binding to phenylhydrazine-treated human erythrocytes: implications for models of red blood cell aging.

A fundamental difficulty in the study of red cell senescence has been that of isolating a population of cells that have been aged in vivo. It has been proposed that the induction of Heinz body formation through the use of oxidizing agents, including phenylhydrazine, can provide a model for elucidating mechanisms of normal red cell aging. In an effort to evaluate the applicability of this model, we examined the nature of autologous antibody binding to phenylhydrazine-treated cells. Using both radioisotopic and immunofluorescent probes, we confirmed the previous observation of increased immunoglobulin G (IgG) binding to phenylhydrazine-treated cells. However, further analysis of the cell population indicated that the majority of the IgG bound to cells that had suffered severe membrane lesions, allowing access to intracellular consituents. Elution and analysis of the bound autologous IgG confirmed that it showed specificity for some of these intracellular constituents as well as for the surface epitopes available on intact cells. We conclude that phenylhydrazine treatment is a problematic model for study of red blood cell aging mechanisms, because the binding of autologous IgG appears to be limited to a small population of severely damaged cells.

Comparison of serum anti-band 3 and anti-Gal antibody binding to density-separated human red blood cells.

This study examines the quantitative relationship between two natural serum antibodies, anti-band 3 and anti-alpha-galactosyl (anti-Gal), in their capacity to bind to human red blood cell (RBC) populations separated on density gradients. The question was approached in two ways. First, we determined the extent of rebinding of affinity-purified human serum antibodies to RBCs that had been stripped of in situ antibody. Second, we eluted the in situ bound antibody at low pH from density-separated RBCs and determined the proportion of total eluted antibody that bound specifically to erythrocyte band 3 or to a Gal-alpha-(1,3)-Gal structure. Our results show that high-density human RBCs bind increased amounts of both antibodies. Anti-Gal rebinding was specific, because it was saturable and occurred in the presence of serum IgG depleted of anti-Gal. Binding assays using control natural autoantibodies directed against antigens not found on the RBC surface showed that high-density RBCs also bind increased amounts of these antibodies as compared with low-density RBCs. However, the extent of this binding is substantially lower than that of anti-band 3 and anti-Gal. Binding studies using the lectins Bandeiraea Simplicifolia (alpha-galactosyl specific) and Arachis Hypogaea (peanut agglutinin, beta-galactosyl specific) indicated that only the alpha-galactosyl sites are exposed on high density RBCs, and not the beta-galactosyl structure characteristic of T antigen. Antibody that is eluted at low pH from high density RBCs contains a 5.0% to 18.0% component that binds to band 3 protein, and a 9.1% to 39.0% component that recognizes the alpha-galactosyl structure. Together, the two antibodies appear to constitute an average of 35% (range 17.2% to 57.4%) of the in situ bound antibody from high-density human RBCs.

Ektacytometric measurement of sickle cell deformability as a continuous function of oxygen tension.

In an effort to study the rheologic effects of small amounts of hemoglobin S (HbS) polymer in sickle red cells, we have used the ektacytometer, a laser diffraction couette viscometer, to measure sickle cell deformability as a function of oxygen tension. Sickle cell populations of defined intracellular hemoglobin concentration (MCHC) were isolated using Stractan density gradients and were resuspended in buffered polyvinylpyrrolidone solutions for deformability measurements. Using a gas-porous, hollow fiber gas exchange system to establish a linear gradient in oxygen tension, deformability was measured over a pO2 range of 76 to 0 mm Hg. Parallel spectroscopic determinations of oxygen saturation permitted determination of cell deformability as a function of oxygen saturation for each discrete MCHC population. From these measurements the level of oxygen saturation at which a loss in cell deformability was first detected could be defined. Then, using the data of Noguchi and Schecter, the amount of polymerized HbS in the cells at that defined level of oxygen saturation was estimated. The results of this analysis suggested that the quantity of polymer that caused a detectable loss in cell deformability increased with increasing MCHC. In addition, for MCHC above 30 g/dL, this represented a substantial fraction of the total HbS in the cell.

Host-associated iron transfer factor in normal humans and patients with transfusion siderosis.

A low molecular weight iron-binding substance that promotes bacterial growth in vitro by increasing iron availability was identified in human blood and urine. Partial purification and physical characterization indicate that this factor is similar to the host-associated iron transfer factor (HAITF) previously isolated from mammalian tissue. HAITF was found to be significantly elevated in the blood of patients with thalassemia who have transfusional siderosis. The level of HAITF in the blood of these patients was also found to correlate with that of serum iron and serum glutamic-oxaloacetic transaminase (SGOT) but not with that of serum ferritin. Thus, elevated blood levels of HAITF may explain the increased susceptibility to infection seen in patients with iron overload. Its physiologic role, however, may involve the transport of iron within cells.

AUR memorial Award. Induced alignment of flowing sickle erythrocytes in a magnetic field. A preliminary report.

Deoxygenated sickle erythrocytes in static suspension align perpendicular to a magnetic field. To assess the importance of this observation to MRI of sickle-cell disease, an in vitro flow apparatus was devised and the orientation of sickle erythrocytes flowing through a 0.38 T magnetic field was investigated. We showed a significant perpendicular alignment of fully deoxygenated sickle erythrocytes flowing at 3 to 4 mm/minute (P less than .001). These results suggest that deoxygenated erythrocytes in a sickle-cell patient could orient perpendicular to a magnetic field, and therefore that MRI of such patients could possibly result in worsening of vaso-occlusive complications. Further studies are needed to assess the possible hazards of MRI of sickle-cell disease, especially at high field strengths.