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Toward a new reference method for the leukocyte five-part differential

W Hübl1, G Wolfbauer, S Andert

  • 1Central Laboratory, Wilhelminenspital, Vienna, Austria.

Cytometry
|April 15, 1997
PubMed
Summary
This summary is machine-generated.

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This study validates a flow cytometry method for leukocyte differential counts, proposing it as a new automated reference for basophil counting. While accurate for basophils, other cell counts showed variability due to sample preparation and flow cytometer differences.

Area of Science:

  • Clinical immunology
  • Hematology
  • Flow cytometry

Background:

  • Accurate leukocyte differential counts are crucial for diagnosing and monitoring various hematological conditions.
  • Existing manual methods are labor-intensive and prone to variability.
  • Development of automated, precise, and reproducible methods is essential for clinical laboratories.

Purpose of the Study:

  • To evaluate a novel three-color flow cytometric method for a five-part leukocyte differential.
  • To assess the accuracy and reproducibility of the flow cytometry method against manual differentials.
  • To identify factors influencing flow cytometric results, including sample preparation, instrument variation, and storage.

Main Methods:

  • A five-part leukocyte differential was performed using flow cytometry with anti-CD45, anti-CD14, anti-CD2, anti-CD16, and anti-HLA-DR antibodies.

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  • Three distinct sample preparation procedures and two different flow cytometers were utilized.
  • Results were compared with a 1,000-cell manual differential count for accuracy assessment.

    • Main Results:

    • Excellent correlation was observed between flow cytometry and manual differentials for most leukocyte subclasses.
    • Flow cytometric basophil counts showed perfect agreement with manual counts across all tested conditions.
    • Sample preparation and flow cytometer type influenced results for other leukocyte subclasses, though cell loss was not a factor.
    • Minimal changes in results were noted within 24 hours of sample storage.
    • A significantly higher monocyte count was detected in males using the flow cytometry method.

    Conclusions:

    • The evaluated flow cytometry method is proposed as the first acceptable automated reference method for basophil counting.
    • Methodological standardization, particularly concerning sample preparation, is critical for improving interlaboratory reproducibility for other leukocyte subsets.
    • Flow cytometry offers a precise and suitable reference method for five-part leukocyte differentials, with potential for improved diagnostic accuracy.