Evaluation of a new complement‐dependent lymphocytotoxicity cross match method using an automated cell counter, the NucleoCounter® NC‐3000™

Abstract

Complement‐dependent lymphocytotoxicity cross match (CDC‐XM) is the ultimate test of donor/recipient compatibility prior to organ transplantation. This test is based on cell viability, evaluated under fluorescence microscopy by an operator after proper staining. The determination of the positivity threshold may vary depending on the operator. We developed a new method in which the final step of determining cell viability is automated using the NC‐3000™ (Chemometec®), an image cytometer able to precisely determine the percentage of dead/live cells in a suspension. After T and B donor cells isolation by negative selection, complement‐dependent lysis was performed in macrovolumes in a PCR plate. Then, cell viability was measured by the NC‐3000™. The sensitivity and routine CDC‐XM results of this new method were compared to those of CDC‐XM reference method using Terasaki plates. The sensitivity of CDC‐XM expressed in the ASHI scoring system of this method was similar to the reference method results for a dilution range of the positive controls. Similarly, the results of the new method were comparable in a clinical situation to those obtained with the reference method after a study of 10 cross‐matches, of which 5 cross‐matches with DSA were positive and five cross‐matches without DSA were negative. Moreover, ASHI scores were similar to those obtained using the reference method, and the mortality percentage was reproducible (CV < 15%). The assessment of cell viability by the NC‐3000™ is easy to perform and highly reproducible but requires CDC‐XM to be performed by the macrovolume method. The determination of a precise percentage of viability/mortality by the automation excludes operator variability and allows a better understanding of results close to the decision threshold.