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.
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.
Schmidtke
G
G
Department of Internal Medicine, Otto-von-Guericke-University of Magdeburg, Germany.
Schmucker
U
U
Brittinger
G
G
Höffkes
H G
HG
D000368:Aged; D000911:Antibodies, Monoclonal / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology / Q000473:pathology*; D001793:Blood Preservation; D002454:Cell Differentiation; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D013194:Staining and Labeling / Q000379:methods
D000911:Antibodies, Monoclonal