Background:

Flow cytometric immune-phenotyping is influenced by cryopreservation and inter-laboratory variability limiting comparability in multi center studies. The Duraclone™ system (DC) provides optimized pre-mixed dry antibody panel tubes for use with small amounts of whole blood (1ml for 6 panels) and a standardized reading protocol. We assessed DC for validity, reliability and challenges when used in a multicenter study with long distance sample shipping.

Methods:

Within the POSITIVE study, a Canadian multicenter collaboration (CNTRP) 38 children awaiting transplant (TX, 24 kidney, 8 liver, 6 heart) were enrolled for parallel immune-phenotyping with DC and validated, optimized in-house panels (ST). Samples were collected before, 3 and 12 months post-Tx. Quality assurance measures and congruence of phenotypes were compared considering age groups, organ types and clinical factors using Bland-Altman comparisons, linear regression and group comparisons.

Results:

Both assays showed excellent viability (mean 94.8%) and lymphocyte recovery with no significant decrease when processed within 30h. Recovery was lower and more dependent on time to processing in post-Tx samples on immune suppression. Comparing ST and DC mean difference (<5%) and range of deviation (2SD <15%) were found for T-cells: CD4+ (-3.8%), CD8 (+4.1%) and regulatory CD4+ T-cells (-2.5%), with variation 2SD<20% for B-cells CD19 (+1.7%), and NK-cells CD56 (+4.1%), and highly significant correlation between methods (p<0.001). Variation of 2SD <25% was found for CD27+IgM- switched memory B-cells (-2.1%) and CD24hCD38h transitional B-cells (+3.4%) when detected by population gate. Systematic under-detection was found in DC for CD27-CD28- 'exhausted' T-cells and for plasma blasts (-35%).

Conclusions:

The DC system provides a reliable method for standardized analysis of many immune phenotypes after long distance shipping when processed within 30h. Some phenotypes are under-detected and plasma blasts appear to decline, possibly due to red cell lysis or EDTA preservation. Within these limitations DC is attractive for pediatric studies due to small amounts of blood required and highly standardized processing and analysis.

CITATION INFORMATION: Urschel S., Ionescu L., Blydt-Hansen T., Foster B., Mital S., Allan U., Iveson S., Hamiwka L., Yap J., Phan V., Birk P., West L., Levings M. Immune Phenotyping in a Pediatric Multicenter Transplant Study: Suitability of a Pre-Formulated Dry Antibody Panel System Am J Transplant. 2017;17 (suppl 3).

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