Time from venipuncture to cell isolation: Impact on granulocyte-reactive antibody testing

Background and objectives: Classical neutrophil-reactive antibody testing depends on the quick isolation of neutrophils from freshly taken whole blood. To allow a better logistic preparation before testing, the influence of time interval between venipuncture and cell isolation has been evaluated in this study.

Materials and methods: Neutrophils and whole leukocytes were isolated from EDTA whole blood immediately (T0) as well as 4, 8 and 24 h after blood donation (T4, T8 and T24). These cells were tested against reference sera containing antibodies against HNA-1b, -2, -3a and HLA class I using granulocyte aggregation test (GAT), microscopic granulocyte immunofluorescence test (GIFT) and flow-cytometric white blood cell immunofluorescence test (Flow-GIFT/WIFT).

Results: GAT was the most error-prone test displaying overall weaker aggregation strengths already at T4 (overall accuracy OA = 0.72, κ = 0.58). GIFT results showed good agreement at T4 (OA = 0.86, κ = 0.79) and remained stable until T8, while test results were slightly impaired at T24 (OA = 0.71, κ = 0.55). Flow-GIFT/WIFT was identified as the most robust screening method, remaining stable even at T24. Calculated ratios (sample/negative control) decreased non-significantly and remained highly above the cut-off in all samples.

Conclusion: Acceptable time limits for cell isolation are different for each screening method investigated. For GAT, cell isolation should be performed within 4 h, while GIFT tolerates a neutrophil isolation delay of 8 h. Flow-GIFT/WIFT isolation can be performed even after 24 h without impairment of the results. Using the latter test as a stand-alone pre-screening test, whole blood can be used from donors who are not directly accessible.