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

Influence of Pre-Analytic Conditions on Quantity of Lymphocytes.

Author(s): Proschmann U, Shalchi Amirkhiz P, Andres P, Haase R, Inojosa H, Ziemssen T, Akgün K

Publication: Int J Mol Sci, 2023, Vol. 24, Page

PubMed ID: 37686285 PubMed Review Paper? No

Purpose of Paper

This paper compared immune cell counts in matched blood specimens that were collected from volunteers in different body positions at the time of specimen collection (sitting, supine, standing), after different tourniquet times (10, 30, 150 s), and using different blood aspiration speeds (60, 16–20, and 10–15 s).  Immune cell counts were also assessed before and after blood storage at room temperature, 4°C, and 37°C for up to 24 h.

Conclusion of Paper

There was no effect of tourniquet time, patient posture, or aspiration speed on the counts of immune cell subsets. Similarly, storage of blood at 4°C or room temperature for ≤ 24 h had no effect on the counts of most immune cell types, although significantly fewer activated CD3+ T cells were observed after blood storage for 24 h at 4°C or room temperature. Storage of blood at 37°C for 4 h resulted in a significant decrease in the absolute cell count of CD19+ B cells and, after 24 h, significant declines in lymphocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells, activated CD3+ T cells, and monocytes.

Studies

  1. Study Purpose

    This study compared immune cell counts in matched blood specimens that were collected from volunteers in different body positions at the time of specimen collection (sitting, supine, and standing), after different tourniquet times (10, 30, 150 s), and using different blood aspiration speeds (60, 16–20, and 10–15 s). Immune cell counts were also assessed before and after blood storage at room temperature, 4°C, and 37°C for up to 24 h. Blood was collected from twenty healthy donors (five per experiment) in EDTA tubes using a 21-gauge needle. To investigate the effect of patient posture, blood was collected from 5 volunteers (one female and four males) when they were sitting, supine, and standing; there was a 10 min interval between a postural change and blood draw. To investigate the effects of tourniquet time, blood was collected from five volunteers (three females and two males) after tourniquet placement for 10 s, 30 s, and 150 s (tourniquet time). To investigate the effects of aspiration speed during the blood draw, blood from five volunteers (three females and two males) was aspirated at a slow (max 60 s), medium (16–20 s), and fast (10–15 s) speed. To investigate the effects of delayed processing, blood from five patients (three females and two males) was stored at room temperature, 4°C, and 37°C for 0, 4, and 24 h before immunophenotyping. Unless otherwise specified, blood was immediately diluted in 3% acetic acid solution and cells were counted in triplicate in a Neubauer Chamber. Cells were stained using antibodies against CD45, CD3, CD19, CD8, CD4, CD14, and CD56 and counted on a LSR Fortessa cytometer.

    Summary of Findings:

    There were no effects of tourniquet time, patient posture, or aspiration speed on the counts of immune cell subsets. Similarly, storage of blood at 4°C or room temperature for ≤ 24 h did not affect counts of lymphocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells, monocytes, or granulocytes, although storage at room temperature for 24 h resulted in significantly fewer activated CD3+ T cells (P=0.029 and P=0.02, respectively). Storage of blood at 37°C for 4 h resulted in a significant decrease in absolute CD19+ B cell counts (P=0.02); longer delays for 24 h resulted in significant declines in lymphocytes (P=0.001), CD3+ T cells (P=0.005), CD4+ T cells (P=0.029), CD8+ T cells (P=0.001), activated CD3+ T cells (P=0.018), and monocytes (P=0.018), but not granulocytes or natural killer cell counts.

    Biospecimens
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    Cell count/volume Flow cytometry
    Cell count/volume Light microscopy
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Acquisition Patient posture Sitting
    Supine
    Standing
    Storage Storage temperature Room temperature
    4°C
    37°C
    Storage Storage duration 0 h
    4 h
    24 h
    Biospecimen Acquisition Method of fluid acquisition Aspiration rates compared
    Tourniquet times compared

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