Why is it relevant to perform an endothelial cell crossmatch in pre-transplant evaluations?

Despite extensive testing before renal transplantation, a large proportion of transplanted patients experience rejection. Even though negative lymphocyte crossmatch (LXM) tests indicate a lack of antibodies against HLA class I and II, many patients experience rejection episodes that might result in graft loss or decreased graft function. The clinical data in our study show a strong correlation between the presence of anti-endothelial cell antibodies and rejection episodes - despite the negative LXM.

What correlation has been observed between lymphocyte crossmatch (LXM) and Olerup XM-ONE?

The majority of patients included in the study were approved for transplantation based on negative LXM. However, 14 patients were still ‘considered safe' for transplantation despite being LXM positive. Reasons for this decision included lack of donor-specific antibodies or an inconsistent LXM result. In the multi-center study, none of the patients with a positive flow LXM and a concomitant negative Olerup XM-ONE experienced antibody-mediated rejection.

Is there a correlation between anti-endothelial cell antibodies and long-term kidney function?

All antibody-mediated rejections occurred in Olerup XM-ONE positive patients. Biopsies from the first rejection episode showed that 6 patients were C4d positive. All were in the Olerup XM-ONE positive group. Several studies have shown a strong correlation between severity of rejection episode and graft outcome. 
For mean creatinine values 3 and 6 months after transplantation, a significant difference between Olerup XM-ONE positive and negative patients was observed. Data have been published showing a strong correlation between post-transplant creatinine value and long-term graft survival.

How specific are the anti-endothelial cell antibodies? If a patient is positive against one donor, what is the probability for also being positive against a pool?

The probability that a patient who is positive against one individual would also be positive against a pool is relatively high, but not 100%. We have seen patients with graft losses and endothelial cell antibodies against only the donor and no reaction against other potential donors.

Can Olerup XM-ONE be used for crossmatch tests in deceased donors?

Yes, as long as you can sample peripheral blood, Olerup XM-ONE can be used. In the study, 33 of the 147 patients received kidneys from deceased donors. The result in this sub-group correlates well with the total study.

What is the experience of auto-antibody testing with Olerup XM-ONE?

Although we did not include auto-antibody tests in the study protocol, a few mandatory cases were performed. In 3 patients with positive Olerup XM-ONE, we also found auto-antibodies against endothelial cells. None of these 3 had any rejection episodes.



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 Olerup 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 kit.

What cut-off values should be used for Olerup 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.