Use of automated differential counter for thawed samples

Dear Flow-ers,

I am in the process of validating our freezing protocol for cord blood samples. To assess our nucleated cell recovery post thaw, I am using a Coulter A(c).T diff Analyzer. However there is a 4-5 fold difference in the manual counts compared to the coulter counts. Our counter is well calibrated and counts fresh cord blood samples accurately. The difference between the coulter and manual counts is <10% for fresh samples. Therefore my guess is that the clumps and debris in the thawed samples, is throwing the coulter off. Since I have the coulter counts of the fresh samples, I want to use the same platform for counting cells post thaw to accurately determine nucleated cell recovery.

I am sure somebody out there has faced a similar problem. If so, I would like to know what is routinely done in such cases.

Thanks

Reliance Life Sciences

Haematopoietic Stem Cell Lab

5th floor, Tower Building

Sir HN Hospital

Raja Ram Mohan Roy Road

Mumbai 400004

India