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

SCHMIDTKE, G.
SCHMUCKER, U.
BRITTINGER, G.
HÖFFKES, H.-G.

Division of Haematology/Oncology, Department of Internal Medicine, Otto-von-Guericke-University of Magdeburg, Germany and *Division of Haematology, Department of Internal Medicine, University of Essen, Germany

Accepted for publication 9 September 1998

Correspondence: Dr H.-G. Höffkes, Division of Haematology/Oncology, Department of Internal Medicine, Otto-von-Guericke-University of Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany.

Clinical & Laboratory Haematology 21(2):p 103-112, April 1999.

Summary

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 °C, 24 °C and 30 °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 °C and 24 °C but immunoglobulin staining showed best results when blood was stored at 4 °C. Storage at 30 °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 °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.