title: Relevance of flow cytometric enumeration of post-thaw leucocytes: influence of temperature during cell staining on viable cell recovery.
abstract: OBJECTIVE
  Our post-thaw cell recovery rates differed substantially in interlaboratory comparisons of identical samples, potentially due to different temperatures during cell staining.
  
  
  METHODS
  Viable CD34(+) cells and leucocyte (WBC) subtypes were quantified by multiparameter single-platform flow cytometry in leucapheresis products collected from 30 adult lymphoma and myeloma patients, and from 10 paediatric patients. After thawing, cells were prepared for analysis within 30 min between thawing and acquisition, at either 4°C or at room temperature.
  
  
  RESULTS
  For cell products cryopreserved in conventional freezing medium (10% final DMSO), viable cell recovery was clearly lower after staining at 4°C than at RT. Of all WBC subtypes analysed, CD4(+) T cells showed the lowest median recovery of 4% (4°C) vs. 25% (RT), followed by CD3, CD34 and CD8 cells. The recovery was highest for CD3γδ cells with 44% (4°C) vs. 71% (RT). In the 10 samples cryopreserved in synthetic freezing medium (5% final DMSO), median recovery rates were 89% for viable CD34 (both at 4°C and RT) and 79% (4°C) vs 68% (RT) for WBC.
  
  
  CONCLUSIONS
  The post-thaw environment and, potentially, the cryoprotectant impact the outcome of cell enumeration, and results from the analysis tube may not be representative of the cells infused into a patient.
authors.affiliation: Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: G
authors.initials: G
authors.lastname: Fritsch
authors.affiliation: Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: N
authors.initials: N
authors.lastname: Frank
authors.affiliation: Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: J
authors.initials: J
authors.lastname: Dmytrus
authors.affiliation: Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: C
authors.initials: C
authors.lastname: Frech
authors.affiliation: St. Anna Kinderspital, Universitätskinderklinik, Vienna, Austria.
authors.forename: H
authors.initials: H
authors.lastname: Pichler
authors.affiliation: St. Anna Kinderspital, Universitätskinderklinik, Vienna, Austria.
authors.forename: V
authors.initials: V
authors.lastname: Witt
authors.affiliation: Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: R
authors.initials: R
authors.lastname: Geyeregger
authors.affiliation: Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: D
authors.initials: D
authors.lastname: Scharner
authors.affiliation: Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: D
authors.initials: D
authors.lastname: Trbojevic
authors.affiliation: Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: E
authors.initials: E
authors.lastname: Zipperer
authors.affiliation: Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: D
authors.initials: D
authors.lastname: Printz
authors.affiliation: Dept. for Blood Group Serology and Transfusion Medicine, Medical University, Vienna, Austria.
authors.forename: N
authors.initials: N
authors.lastname: Worel
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000378:metabolism; D005434:Flow Cytometry; D005615:Freezing; D006801:Humans; D007962:Leukocytes / Q000166:cytology* / Q000378:metabolism; D009101:Multiple Myeloma; D013194:Staining and Labeling; D013696:Temperature
chemical_list: D018952:Antigens, CD34
keywords: CryoStor CS10; DMSO; cell loss; cell recovery; cell viability; post-thaw cell enumeration