RU2547573C2 - Method for detecting phosphatidyl serine on erythrocyte membrane surface - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biology and medicine, namely to laboratory methods of examination. Detecting phosphatidyl serine on the erythrocyte membrane surface is ensured by fixing cells by glutaric aldehyde, washing in distilled water and placing on a slide processed with lanthanum chloride, mixing and studying cell aggregation by optical microscopy. Observing the erythrocyte aggregates testifies to the presence of phosphatidyl serine on the erythrocyte membrane surface.

EFFECT: invention provides developing simple, fast method requiring no special equipment and critical agents and used for detecting phosphatidyl serine on the erythrocyte membrane surface.

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Description

The present invention relates to biology and medicine, namely to laboratory research methods, and can be used to assess the presence of phosphatidylserine on the surface of red blood cell membranes.

In erythrocytes, phospholipids are known to be asymmetrically located. Phosphatidylcholine and sphingomyelin are located on the outer half of the lipid bilayer, phosphatidyl ethanolamine on the outer and inner half of the membrane. Negatively charged phospholipid phosphatidylserine is located exclusively on the inside of the lipid bilayer (Zwaal R.F.A., Schroit A.J. Blood. 1997. Vol.89. P.1121-1132). The release of phosphatidylserine on the surface of blood cell membranes under many pathological conditions accelerates blood coagulation and leads to thrombotic conditions (Zwaal R.F.A, Comfurius P., Bevers E.V. Cell.Mol.Life Sci. 2005. Vol.62. P.971-988).

Lanthanum, being a trivalent cation, has a high affinity for phosphatidylserine and, at low concentrations, selectively interacts with negatively charged phosphatidylserine (Bentz J., Alford O., Cohen J., Duzgunes N. Biophys J. 1988. Vol.53. P.593- 607).

The principle of the proposed method for assessing the presence of phosphatidylserine on the surface of erythrocyte membranes is based on the electrostatic interaction of positively charged lanthanum with negative phosphatidylserine. As a result of the interaction of lanthanum with phosphatidylserine, aggregation of red blood cells occurs.

As a prototype, a method for assessing the presence of phosphatidylserine on the surface of erythrocyte membranes was selected, which includes adding annexin V fluorescence protein to red blood cells and then measuring fluorescence using a flow cytometer (Kuypers FA, Lewis RA, Hua M. Et al. Blood. L996. Vol. 87. Vol99. P.1179-1183).

However, this requires expensive scarce equipment (flow cytometer) and expensive scarce reagents.

The objective of the invention is the improvement of the method.

EFFECT: simplification of the method for assessing the presence of phosphatidylserine on the surface of erythrocyte membranes.

The technical result is achieved due to the fact that in the method? including the fixation of red blood cells with glutaraldehyde, their washing, placement on a glass slide treated with lanthanum chloride, monitoring the appearance of aggregates, the appearance of red blood cell aggregates judges the presence of phosphatidylserine on the surface of red blood cell membranes.

The method is as follows. Red blood cells are fixed in a 1% solution of glutaraldehyde prepared in phosphate buffer for 60 minutes at room temperature. After fixation, red blood cells are washed three times with distilled water, followed by centrifugation at 3000 rpm for 5 minutes. The washed red blood cells are transferred to a glass slide treated with a 0.5 mM lanthanum chloride solution, mixed and observed using a light microscope to form cell aggregates. The appearance of aggregates indicates the presence of phosphatidylserine on the surface of erythrocyte membranes.

It is known that erythrocytes of the krenate form (echinocytes), unlike normal erythrocytes (discocytes), have a high phosphatidylserine content on the membrane surface (Wolfs JLN, Comfurius P., Bevers EM, Swaal RFA Molecular Membrane Biology. 2003. Vol.20. P. 83-91).

Discocytes and echinocytes were used as examples. Echinocytes were obtained by depletion; by ATP (Feo C., Mohandas N. Nature. 1977. Vol.265. P. 166-168) and after treatment with lysophosphatidic acid (Chung SM, Bae ON, Lim KM et al. Arterioscler. Thromb. Vasc. Biol. 2007 Vol.27. P.414-421).

Example 1. Fixed discocytes are placed on a glass slide treated with lanthanum, mixed and observed using a light microscope. There is a lack of red blood cell aggregates. On the surface of the erythrocyte membranes, phosphatidylserine is absent.

Example 2. Fixed echinocytes obtained after depletion by ATP, placed on a glass slide treated with lanthanum, mixed and observed using light microscopy. Red blood cell aggregates are observed. Phosphatidylserine is present on the surface of erythrocyte membranes.

Example 3. Fixed echinocytes obtained after treatment with lysophosphatidic acid, placed on a glass slide treated with lanthanum, mixed and observed using light microscopy. Red blood cell aggregates are observed. Phosphatidylserine is present on the surface of erythrocyte membranes.

Thus, the proposed method for assessing the presence of phosphatidylserine on the surface of erythrocyte membranes does not require special equipment.

The advantage of the proposed method is simplicity, accessibility, speed, the absence of the need for complex expensive equipment and expensive scarce reagents.

Claims (1)

A method for assessing the presence of phosphatidylserine on the surface of erythrocyte membranes, comprising fixing the red blood cells with glutaraldehyde, characterized in that fixed red blood cells are placed on a glass slide treated with lanthanum chloride to mix the presence of phosphatidylserine, and the presence of aggregates is judged on the presence of phosphatidylserine phosphatides on the surface.