Ficoll hypaque density cushion

Immediately prior to surgery, a blood sample was drawn from a central venous line into a lithium heparin monovette (Sarstedt, Nümbrecht, Germany). The bone marrow was punctured at the right iliac crest and 10 mL of bone marrow blood were taken and drawn into a lithium heparin monovette as well. All samples were kept at room temperature (18–25 °C) and were further processed within 0.5–2 h. Separation of the mononuclear cell fraction was performed by centrifugation through a Ficoll-Hypaque density cushion (GE Healthcare, Freiburg, Germany). Mononuclear cell fractions were then isolated, washed in phosphate buffer saline (PBS) and cells were counted in a Neubauer chamber. For RNA preparation, cells were lyzed in PeqGold RNApure™ reagent (PeqLab, Erlangen, Germany) and total RNA was isolated according to the manufacturer’s protocol.

Instantly prior to surgery, a blood sample was drawn from a central venous line into a lithium heparin-Monovette (Sarstedt, Nümbrecht, Germany). All samples were kept at room temperature (18°C-25°C) and were further processed within 0.5-2 hours. Separation of the mononuclear cell (MNC) fraction was performed by centrifugation through a Ficoll-Hypaque density cushion (GE Healthcare, Freiburg, Germany). MNCs were then isolated, washed in PBS and counted.

Prior to surgery, 10 ml bone marrow blood was aspirated from the spina iliaca anterior under general anesthesia subsequent to a small cutaneous incision. Venous blood (20 ml) was taken in parallel from a central venous line. Lithium heparin was used as anti-coagulant. Fractions of mononuclear cells from blood or bone marrow were isolated by centrifugation through a Ficoll-Hypaque density cushion (GE Healthcare, Freiburg, Germany) according to the manufacturer’s recommendation. After washing in PBS, cells were counted, pelleted again, and subsequently centrifuged onto microscopic slides (cytospins) or lysed for RNA preparation with RNAPure reagent (PQLab, Erlangen, Germany) and further processed according to the manufacturer’s protocol. Total RNA was isolated and checked for integrity using a Bioanalyzer 2100 instrument (Agilent Technologies, Böblingen, Germany). CDNA synthesis and nested CK20 RT-PCR analysis was exactly performed as previously described in detail [11 (link)]. Every sample was assessed in triplicate. If at least one positive PCR test out of three was obtained, the sample was rated as CK20-positive. All assessments of PCR results were performed blinded, without knowledge of the clinical data.