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...

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Bibliographic Details
Published in:Clinical and laboratory haematology 1999-04, Vol.21 (2), p.103-112
Main Authors: Schmidtke, G, Schmucker, U. SCHMUCKER, Brittinger, G. BRITTINGER, Höffkes, H.-G.
Format: Article
Language:English
Subjects:
Aged
Antibodies, Monoclonal - immunology
B-Lymphocytes - immunology
B-Lymphocytes - pathology
Biological and medical sciences
Blood Preservation
Cell Differentiation
Chronic lymphocytic leukaemia
Female
Flow Cytometry - methods
Hematology
Humans
immunophenotyping
Immunophenotyping - methods
Investigative techniques, diagnostic techniques (general aspects)
Leukemia, Lymphocytic, Chronic, B-Cell - immunology
Leukemia, Lymphocytic, Chronic, B-Cell - pathology
Male
Medical sciences
Middle Aged
Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques
pre-analysis
Staining and Labeling - methods
storage
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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 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.
ISSN:0141-9854
1365-2257
DOI:10.1046/j.1365-2257.1999.00203.x