Comparative flow cytometric study of clonal excess in leukaemic peripheral blood from patients suffering from chronic lymphocytic leukaemia (B-CLL) by different antibodies, staining techniques and the effects of blood storage

Clin Lab Haematol. 1999 Apr;21(2):103-12. doi: 10.1046/j.1365-2257.1999.00203.x.

Abstract

This investigation studied the effects of cell preparation methods, different antibody panels and blood storage on antigen expression of abnormal B lymphocytes from patients with B-CLL. Blood specimens collected in Heparin de novo were processed by using conventional Hypaque-Ficoll density gradient centrifugation and whole blood lysis. These were stored for 3 days at 4 degrees C, 24 degrees C and 30 degrees C. Although clonal excess was detected by all antibody panels, significant differences could be observed in terms of molecules of equivalent fluorochromes (MEF/MESF units). Evaluation of 'weak and strong' staining is dependent on the antibody panel used. Immunofluorescent values for CD19 and CD45 were unchanged at 4 degrees C and 24 degrees C but immunoglobulin staining showed best results when blood was stored at 4 degrees C. Storage at 30 degrees C produced unreliable results. Abnormal B lymphocytes should be analysed immediately after the specimen is obtained. If shipment is necessary they should be kept at 4 degrees C. Surface immunoglobulins are the 'antigens' most sensitive to storage alterations. Sample alterations alone are sufficient to the correct classification of NHL, especially in the case of low-grade NHL.

Publication types

  • Comparative Study

MeSH terms

  • Aged
  • Antibodies, Monoclonal / immunology
  • B-Lymphocytes / immunology
  • B-Lymphocytes / pathology*
  • Blood Preservation
  • Cell Differentiation
  • Female
  • Flow Cytometry / methods
  • Humans
  • Immunophenotyping / methods*
  • Leukemia, Lymphocytic, Chronic, B-Cell / immunology
  • Leukemia, Lymphocytic, Chronic, B-Cell / pathology*
  • Male
  • Middle Aged
  • Staining and Labeling / methods

Substances

  • Antibodies, Monoclonal