Polymorphprep™ is a ready-to-use, sterile, endotoxin-tested separation medium optimized for the isolation of human granulocytes (polymorphonuclear leukocytes, PMNs). After centrifugation of undiluted human whole blood samples through Polymorphprep™, PMNs accumulate in a distinct band above the pelleted erythrocytes and below the retained mononuclear cells at the sample/medium interface.
- Density 1.113 ± 0.001 g/ml
- Osmolality 445 ± 15 mOsm
- Endotoxin level < 1.0 EU/ml
- Sodium diatrizoate 13.8% (w/v)
- Dextran 500 8.0% (w/v)
In principle, the higher buoyant density (>1.085 µg/ml) of PMNs (with the exception of basophils) can be exploited for their isolation from lighter mononuclear cells (<1.077 g/ml). However, the high buoyant density of erythrocytes (1.09-1.11 µg/mL) complicates the separation of whole blood using density gradient media such as those used for mononuclear cells. However, the high osmolality of Polymorphprep™ causes erythrocytes to lose water and shrink, increasing their effective buoyant density, and allowing dextran-added erythrocytes to rapidly sediment through the dense medium.
Because the osmotic gradient between the medium and the erythrocytes decreases as the cells sediment further into the medium (ie, the loss of water from the erythrocytes is greatest at the top of the gradient and decreases progressively with sedimentation), a diatrizoate gradient forms within the density mattress. Within this gradient, PMNs accumulate in a distinct band below mononuclear cells that are retained at the sample/medium interface. The method is effective only with undiluted whole blood, not with a leukocyte-rich fraction.
Temperature affects the density and viscosity of Polymorphprep™ solutions. Therefore, the temperature of blood samples and media should be maintained between 18 and 22 °C. The following figure shows a Coulter STKR Cell Analyzer analysis of the upper and lower bands of a Polymorphprep™ separation, presented as the relative number of cells (ordinate) as a function of cell volume (abscissa). Relative cell number is the number of cells expressed as a fraction of the total in each sample. The upper band contains only lymphocytes (40-80 femtoliters) and monocytes (80-130 femtoliters). All PMN (150-320 femtoliters) accumulate in the lower band, with insignificant contamination of mononuclear cells and erythrocytes between 2-6% of the total number of cells.