Generation of annexin V-positive platelets and shedding of microparticles with stimulus-dependent procoagulant activity during storage of platelets at 4 degrees C.

BACKGROUND: The ability of propyl gallate to activate platelet factor 3 has been determined through the activated partial thromboplastin time, but its effect on phosphatidylserine has not been established. STUDY DESIGN AND METHODS: A novel platelet activator, propyl gallate, was introduced to a study of platelets stored at 4 degrees C. The effects of storage on platelet coagulation activity, on phosphatidylserine, and on the shedding of activated and activable membrane particles (microparticles) were examined by activated plasma clotting time, and the effect on annexin V binding was examined by gated flow cytometry. The ratios of annexin V binding and microparticle shedding in stored platelet samples were compared with those in fresh platelets stimulated with propyl gallate. RESULTS: Microparticle shedding by stored platelets compensated for the diminished procoagulant potential of intact platelets (shown as the total propyl gallate-dependent platelet factor 3 activity), which did not change during prolonged (20-day) storage, but levels of phosphatidylserine confined to microparticles increased dramatically as platelet counts fell. Both annexin V binding and microparticle shedding increased spontaneously with storage and artificially with propyl gallate stimulation. However, at the same level of annexin V binding, stored platelets shed more microparticles than did fresh platelets stimulated with propyl gallate. CONCLUSION: Propyl gallate induces platelet procoagulant activity and annexin V binding. Stored platelets differ from fresh platelets in a lower reactivity to propyl gallate activation and a higher rate of microparticle shedding.

An optically clear hypercholesterolemic hypertriglyceridemic quality-control material prepared from animal lipid sources.

A hyperlipidemic control serum can be simple prepared from animal lipid sources. Beta- and pre-beta-lipoproteins containing cholesterol and triglyceride are removed from porcine serum by treatment with dextran sulfate and calcium ions. A triglyceride-rich fraction containing only trace amounts of cholesterol is isolated from chicken egg-yolks. The two fractions are then combined in 40 mmol/L sodium bicarbonate to give the desired values for cholesterol and triglyceride. The preparation is stabilized against surface denaturation during long-term storage at 5 degrees C perhaps for as long as two years, by adding 0.25 g of Triton X-100 surfactant per liter, and against an accidental exposure to short-term freezing by adding 10 g of sucrose per liter. We used this solution as a diluent to reconstitute lyophilized bovine serum. The resulting product, having been prepared from only animal sources, is free of hepatitis-associated constituents, and is remarkably clear, homogeneous, and stable. Results obtained with it are precise.

Preparation of lyophilized abnormal hemoglobin control for cellulose acetate electrophoresis.

A process for the preparation of stable lyophilized normal and abnormal hemoglobin controls for alkaline cellulose acetate electrophoresis is described. The hemoglobins were converted to cyanmethemoglobin derivatives with K3Fe(CN)6 and KCN. Sucrose, 100 g/l, was added to stabilize the preparation for lyophilization and storage and to increase the rate of reconstitution. Lyophilized preparations were stable with no loss of resolution for nine months of use in the laboratory and for two weeks of accelerated aging at 37 C. The materials appeared to have an extended shelf-life of at least two years at 5 C. The controls reconstituted quickly, usually within a minute, and could be stored at 5 C for three months with only a slight increase in band streaking. The lyophilized products appeared to be suitable to assist in the development of a quality-control system in hemoglobin screening programs employing cellulose acetate electrophoresis.

A lyophilized hemoglobin control prepared from stroma-free hemolysates.

We prepared a stable lyophilized hemoglobin control from human erythrocytes by removing stroma with calcium and Ficoll. Addition of polyhydroxy compounds enhanced reconstitution and stability. The lyophilized product was stable during one year of use in a service laboratory. Accelerated aging studies demonstrated the potential of up to two years of stability, with methemoglobin increasing at a projected rate of approximately 6% per year. Reconstituted, the control gave reproducible values for at least five days when refrigerated.

Development of a stable lipoprotein diluent for use in reconstituting lyophilized human serum for the preparation of clear, hyperlipidemic quality-control materials.

We describe a simple system for the preparation of a hyperlipidemic control serum. Beta- and pre-beta-lipoproteins are removed from the serum and prepared as a stable diluent; the extracted serum is then lyophilized. Upon addition of the lipoprotein diluent, which was observed to contain only liproprotein and sodium bicarbonate, the serum reconstitutes rapidly, usually within 5 min. By a suitable adjustment of the lipoprotein concentration in the diluent, a hyperlipidemic control serum may be produced with desired concentrations of lipids. Because the lipoproteins are not included in the destructive lyophilization step, the resulting product has remarkable clarity, precision, and stability.

A water-soluble cholesterol derivative for use in augmenting serum control materials.

Cholesterol determination has been hampered by the lack of suitable means to prepare lyophilized serum control materials with an appropriate range of values. We prepared a water-soluble cholesterol derivative by first esterifying cholesterol with adipic acid and then reacting the cholesterol hemiadipate with Polyethylene Glycol 600 to form polyethoxyethanyl-cholesteryl adipate. The compound reacts quantitatively in several commonly used methods, including enzymic, extraction, and direct-assay procedures. When the additive is directly mixed with human serum that has been depleted of beta- and pre-beta-lipoproteins, an optically clear solution results for which cholesterol values are stable. The clarity is retained upon lyophilization and reconstitution. Addition of this cholesterol compound to partially delipidized serum appeared to have no significant effect on results of assay of 18 other commonly measured serum constituents.

Use of a cholesterol-rich bovine lipoprotein to enhance cholesterol concentrations in the preparation of serum control materials.

We describe a process for preparing a cholesterol-rich lipoprotein extract from bovine serum. The material is used to enhance cholestrol concentration up to 3.5 g/liter in a clear control material from human serum. The optical clarity of the enriched serum is only slightly affected by lyophilization and reconstitution. The lipoprotein additive neither produces any evidnet interference in the measurement of the commonly analyzed consitutents nor affects the stability of components after lyophilization and reconstitution.

Addition of triglyceride to serum for use in quality control and reference.

We describe a simple method in which a water-soluble mixture of triocatanoin and a surfactant, "Triton X-114," are used in preparing solutions of triglycerides (triacylglycerols) in either human serum, solutions of albumin, or water. Analytical recovery added triglyceride was quantitative by two methods. The addition did not affect results of analyses for 18 other commonly measured constitutents of serum. When the triglyceride was added to either lipid-depleted human serum or bovine serum albumin solution and lyophilized, subsequent solutions were clear. The triglyceride/protein preparation was stable in lyophilized form for a year and in reconstituted serum for five days at 5 degrees C. Aquenous solutions appear to be stable indefinitely at room temperature.

The use of o-dianisidine for serum haptoglobin electrophoresis using cellulose acetate.

1. Due to the carcinogenicity of benzidine, a method by which o-dianisidine is used to stain serum haptoglobin is described. Serum haptoglobin is determined by electrophoresis using cellulose acetate as the medium. 2. A comparison of the two staining systems demonstrates good agreement. 0-Dianisidine can be substituted for benzidine without loss of specificity.

Preparation of optically clear lyophilized human serum for use in preparing control material.

We describe a simple, rapid process--which includes the specific precipitation of pre-beta and beta-lipoproteins with dextran sulfate and divalent metal ions--for preparing an optically clear human serum that retains its clarity upon reconstitution with water after having been frozen and lyophilized. Such serum contains the normal constituents of human serum, except for the removed lipoproteins. The process causes no apparent interference with results of analyses for 22 of the more commonly measured constituents. Fresh or aged pooled serum or blood-bank plasma containing acid-citrate-dextrose or citrate-phosphate-dextrose are equally suitable as raw materials. This stabilized serum is an excellent matrix for use in preparing standards and quality-control material for assay of components of human serum.