Laboratory-related questions

• What kind of magnet should be used for isolating the endothelial precursor cells?

  Several magnets can be used. We recommend the following magnets Dynal MPC-Lt, DynaMag-15 (sold by Invitrogen) or The Big Easy EasySep Magnet sold by Stem Cell Technologies. Performance of other magnets can not be guaranteed and have to be tested by the performing laboratory. Use of to strong magnets may result I aggregation of beads that can be difficult to dissolve.

• How many endothelial precursor cells could you expect to isolate from a normal healthy adult donor?

  Endothelial precursor cells normally constitute approximately 2% of the PBMC. The actual number will then be determined by the amount of blood cells collected.

• Apart from the tubes being made of silicone or polypropylene, do you recommend tubes from any specific company?

  No, the reason for the above recommendation is that using other tube materials sometimes activates cells, which then adhere to the plastic. Best results are obtained with siliconized glass tubes.

• Is the endothelial precursor cell antigen, to which the bead-conjugated antibody is directed, Tie-2?

  Yes, but surface-staining for Tie-2 after isolation will result in less staining than observed on non-isolated cells because the beads block the surface molecules.

• The way to identify the endothelial precursor cells is by forward side-scatter characteristics. Do you recommend supplementing this method with additional antibody staining?

  No, when using the XM-ONE® kit, we recommend only forward-scatter when performing the assay. If you want to double-check that you have the correct cell population, we recommend using an antibody against CD133, for example. The optimal CD133 antibody would be a monoclonal from RnD (if still available for purchase). Alternatively, you can use a polyclonal antibody instead. During the product development phase, we have performed several tests with relevant surface markers to ensure that we measure the right population with the XM-ONE® kit.

• What cut-off values should be used for XM-ONE®?

  Positive and negative controls are required to interpret the test result. A test sample contains antibodies if it is substantially higher than the negative control. Actual cut-off values depend on the type of equipment used and on the settings. Each testing center has to set its own cut-off values for the XM-ONE® assay, as is the case for all flow cytometry crossmatches performed today.
  
  Example: In a clinical multi-center study performed by AbSorber at five centers using the same equipment, significantly higher rejection frequencies were observed in patients with 50 channel shifts in IgG or 80 channel shifts in IgM compared to the negative control. However, at one center using different equipment, cut-off values were 300 channel shifts for IgG and 400 for IgM.
  
  The result of our clinical study could guide other centers when selecting cut-off values in relation to clinical relevance.

• What does an observation of a negative channel shift mean ie that the patient serum gives a lower value then the negative control?

  A negative channel shift can have many different causes including technical errors. However we have observed that large negative channel shifts (in our studies -80 channels or more) are correlated with poor clinical outcome. The mechanism for this phenomena is unknown but may include prozone effects. We currently recommend diluting the patient serum 1:50 in negative control serum and if the negative channel shift remain or the sample turns positive this should be considered as an indication of increased risk.

• Can lymphocyte crossmatch be performed in the same tube as the endothelial cell crossmatch?

  An ongoing evaluation at Karolinska University Hospital, Huddinge, Sweden, indicates that this might be possible. However, further tests have to be performed to confirm the preliminary results.