Polymorphprep™ is a ready-made, sterile, endotoxin-tested, separation medium optimized for human granulocyte (polymorphonuclear leukocyte, PMN) isolation. Following centrifugation of whole undiluted human blood samples through Polymorphprep™, PMNs accumulate in a distinct band above pelleted erythrocytes and below mononuclear cells retained 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 greater 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 separation from whole blood using density gradient media like that used for mononuclear cells. However, the high osmolality of Polymorphprep™ causes erythrocytes to lose water and shrink, increasing their effective buoyant density, allowing dextran-aggregated erythrocytes to sediment rapidly through the dense medium. Because the osmotic gradient between the medium and erythrocytes declines as cells sediment further into the medium (i.e., water loss from erythrocytes is greatest at the top of the gradient and decreases progressively with sedimentation) – a diatrizoate gradient forms within the density cushion. Within this gradient, PMNs accumulate in a distinct band below mononuclear cells which are retained at the sample/medium interface. The method is effective only from whole undiluted blood, not from a leukocyte-rich fraction.
Temperature impacts the density and viscosity of Polymorphprep™ solutions. Thus, the temperature of blood samples and media should be kept between 18-22°C. Shown in the figure below is a Coulter STKR Cell Analyzer analysis of the top and bottom bands from 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 top band contains only lymphocytes (40-80 femtoliters) and monocytes (80-130 femtoliters). All PMNs (150-320 femtoliters) accumulate in the bottom band with contamination from mononuclear cells being negligible and from erythrocytes between 2-6% of total cell number.