NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials.

Author(s): Ng AA, Lee BT, Teo TS, Poidinger M, Connolly JE

Publication: J Immunol Methods, 2012, Vol. 385, Page 79-89

PubMed ID: 22922462 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of storage, fixative type and shipment at ambient temperatures on cell counts and cell population identification from blood specimens.

Conclusion of Paper

While all cell counts declined during the 10 days of refrigerated storage of unfixed blood, 7 populations declined by more than 50% over the first 4 days of storage and 9 declined by 20% or less. Similarly, mean fluorescence intensity (MFI) of tested markers declined with refrigerated storage, but the magnitude of the decrease depended on storage duration and the marker of interest. Generally, fixation in Cyto-chex was best for maintaining cell counts, and fixation in Transfix was best for maintaining MFI, but paraformaldehyde (PFA) fixation was detrimental to cell counts and population identification. Shipping at ambient temperature in Cyto-chex or Transfix had little effect on cell counts but decreased the ability to identify some cell populations. Shipping unfixed cells at ambient temperature was detrimental to cell counts and ability to identify some cell populations compared to storage of unfixed cells at 4 degrees C.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of storage, fixative type and shipment at ambient temperatures on cell counts and cell population identification from blood specimens. Blood was drawn into EDTA vacutainers and Streck tubes containing Cyto-chex (Streck). EDTA blood was left unpreserved, preserved by the addition of 2 mL Transfix or lysed and preserved with 2 mL of 1% or 4% PFA. Specimens were shipped at ambient temperature round-trip across Singapore and then stored at room temperature until day 10 or stored for the entire duration at 4 degrees C (unfixed or PFA-fixed) or room temperature (Cyto-chex or Transfix-fixed). Cells were stained for 36 different markers.

    Summary of Findings:

    While all cell counts declined during the 10 days of refrigerated storage of unfixed blood, 7 populations declined by more than 50% over the first 4 days of storage and 9 declined by 20% or less. Similarly, the MFI of cell population markers declined with refrigerated storage of unfixed blood, but the magnitude of the decrease depended on storage duration and the marker of interest. Fixative choice impacted different cell types differently. For 5 of the 16 cell types, Cyto-chex-preserved specimens showed fewer changes during storage at room temperature than in unfixed cells at 4 degrees C. Transfix-preserved specimens stored at room temperature maintained 4 of the 16 cell counts better than unfixed cells stored at 4 degrees C, but transfix fixation led to all cells becoming 7AAD positive from day 4 of room temperature storage onward. Specimens fixed in PFA and stored at 4 degrees C did not maintain any cell counts as well as unfixed cells also stored at 4 degrees C. As expected, the MFI of investigated cell population markers were also impacted by fixative choice. Transfix was the best at preserving the identity of cell types, with 8 of the 15 cell types more stable in Transfix-fixed than unfixed specimens. After shipping in Cyto-chex or Transfix at ambient temperature, generally cell counts were comparable to those in similarly fixed but not shipped specimens; however, cell counts of unfixed blood shipped at ambient temperatures were generally lower than in unfixed blood stored at 4 degrees C for a similar duration. Shipping at ambient temperatures in Transfix or Cyto-chex reduced the ability to identify 2 and 3 of the 15 cell populations, respectively, compared to similarly fixed but not shipped specimens that were held at room temperature for a similar duration.

    Biospecimens
    Preservative Types
    • Other Preservative
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    Cell count/volume Flow cytometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 0 days
    4 days
    7 days
    10 days
    Storage Between site transportation method Courier
    Not transported
    Biospecimen Preservation Type of fixation/preservation None (fresh)
    Paraformaldehyde
    Refrigeration
    Cyto-chex
    Transfix
    Biospecimen Preservation Concentration of fixative 1% PFA
    4% PFA
    Storage Storage temperature 4 degrees C
    Room temperature

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